Treatment of retinitis pigmentosa using engineered meganucleases

ABSTRACT

Disclosed are recombinant meganucleases engineered to recognize and cleave recognition sequences present in a mutant RHO P23H allele. The invention further relates to the use of such recombinant meganucleases in methods for treating retinitis pigmentosa, wherein the mutant RHO P23H allele is preferentially targeted, cleaved, and inactivated.

CROSS REFERENCE TO RELATED APPLICATIONS

The application claims the benefit of priority under 35 U.S.C. § 119(e)to co-pending application Ser. No. 62/215,460, filed Sep. 8, 2015, thecontents of which are incorporated in their entirety by reference.

FIELD OF THE INVENTION

The invention relates to the field of molecular biology and recombinantnucleic acid technology. In particular, the invention relates torecombinant meganucleases engineered to recognize and cleave arecognition sequence found in a human rhodopsin gene allele. Theinvention further relates to the use of such recombinant meganucleasesin methods for treating retinitis pigmentosa.

BACKGROUND OF THE INVENTION

Retinitis pigmentosa (RP) is an inherited degenerative eye disease thatcauses severe vision impairment due to the progressive degeneration ofphotoreceptor cells in the retina. RP is characterized by an initialdecline in rod photoreceptor cells, resulting in compromised peripheraland dim light vision. Progressive rod degeneration is followed byabnormalities in the retinal pigment epithelium and deterioration ofcone photoreceptor cells. As the disease advances, patients experiencenyctalopia, progressive tunnel vision, and eventual blindness. RPaffects approximately 1 in 3000 people and can occur alone or togetherwith other systemic disorders. Currently, RP has no effective treatment.

The genetic causes of RP have been identified as autosomal dominant,autosomal recessive, X-linked, or maternally acquired. The autosomaldominant form of RP represents 30-40% of cases (Ma et al. (2105),Scientific Reports. 18(5:9236):1-6), and has been associated withmutations in genes expressed in rod photoreceptor cells and the retinalpigment epithelium. The human rhodopsin gene (RHO) was the first geneshown to contribute to the pathogenesis of autosomal dominant RP andremains the most common gene associated with this form of the disease(McWilliam et al. (1989) Genomics. 5:619-622; Dryj a et al. (1990)Nature. 343:364-366; Farrar et al. (1990) EMBO Journal. 21:857-864).Indeed, RHO mutations are associated with 30-40% of autosomal dominantRP cases worldwide, and are observed in approximately 26.5% of cases inthe United States (Ruing et al. (2002) Journal of Bio. Chem.277(37):34150-34160).

Rhodopsin is an essential photopigment expressed in retinal rodphotoreceptor cells that is responsible for the conversion of lightstimuli into electrical signals in the first step of phototransduction.Rhodopsin is expressed as a light-sensitive G-protein-coupled receptorthat consists of an opsin protein moiety bound to an 11-cis-retinalchromophore, and represents the main component of the disk membranes ofrod photoreceptor cell outer segments.

The first RHO mutation shown to contribute to autosomal dominant RP wasa C to A mutation at position 68 of the RHO gene coding sequence, whichconfers a proline to histidine substitution at position 23 (P23H) of theencoded protein. This mutation is referred to herein as the “RHO P23Hmutation,” and a RHO allele comprising the mutation is referred toherein as a “mutant RHO P23H allele.” The RHO P23H mutation is the mostfrequently reported RHO mutation in autosomal dominant RP cases in NorthAmerica (Mao et al. (2011) Human Gene Therapy. 22:567-575), and patientshaving a single mutant RHO P23H allele can develop RP despite thepresence of a functional wild-type RHO allele.

Rhodopsin proteins that comprise the P23H substitution fold improperly,accumulate in the endoplasmic reticulum of rod photoreceptor cells, anddo not reconstitute with the 11-cis-retinal chromophore. In many casesof autosomal dominant RP, misfolded P23H rhodopsin contributes to rodphotoreceptor cell degeneration and death. Accumulated P23H rhodopsinundergoes proteasomal and lysosomal degradation and has been shown tostimulate the ER-associated unfolded protein response, which can induceER stress and cellular apoptosis (Lin et al. (2007), Science.318:944-949; Gorbatyuk et al. (2010) PNAS U.S.A. 107(13):5961-5966).Misfolding of P23H rhodopsin may also contribute to cell death byinterfering with the transport or function of wild-type rhodopsin(Illing et al., 2002, Lin et al., 2007). Furthermore, P23H rhodopsin hasbeen shown to exhibit delayed dephosphorylation, and cell death mayresult from abnormal cytosolic Ca²⁺ levels (Saito et al. (2008) Clin.Opthamol. 2:821-828).

Multiple strategies have been pursued to treat autosomal dominant RP,including nutritional therapies, pharmaceuticals, and gene therapy. Genetherapy approaches have adopted either an indirect or a direct strategyfor treating autosomal dominant RP. Indirect approaches have aimed topromote the survival of rod photoreceptor cells without directlyaffecting the expression of pathogenic mutant proteins. For example,gene therapy has been used to introduce neurotrophic factors, such asGDNF, and anti-apoptotic proteins, such as XIAP, in retinal cells inorder to inhibit apoptosis in rod photoreceptor cells.

By contrast, direct approaches in gene therapy have sought to modulatethe levels of proteins that directly contribute to the pathogenesis ofautosomal dominant RP. In the context of RHO-associated autosomaldominant RP, one strategy has been to enhance the proteasomaldegradation of P23H rhodopsin, though no significant success has beenmade in animal models. Another strategy has utilized targeted RNA-basedtherapy to silence a mutant RHO allele while maintaining expression ofthe functional wild-type allele. Such approaches have used ribozymes andRNA interference (RNAi) to target specific mRNA transcripts produced bya mutant RHO P23H transgene in rats.

Further strategies have pursued a “suppression and replacement” approachby non-specifically silencing both the wild-type RHO allele and themutant RHO allele, while concurrently delivering a replacement copy ofwild-type RHO to express the wild-type protein. For example, O'Reilly etal. utilized adeno-associated virus (AAV) vectors to deliver and expressshort hairpin RNAs designed to target and suppress both the wild-typeand mutant RHO alleles in heterozygous Pro23His^(+/−) mice, while alsodelivering and expressing a RHO replacement gene (O'Reilly et al. (2007)Amer. J. of Human Genetics. 81:127-135). Palfi et al. similarlydemonstrated the use of AAV vectors to deliver a RHO replacement gene toRho^(−/−) knockout mice (Palfi et al. (2010) Human Gene Therapy.21:311-323). However, in such approaches, toxicity and off-targeteffects may be induced if RHO replacement levels are too high.Furthermore, off-target effects of RNAi approaches are a knowncomplication, and it has been shown that siRNAs greater than 21 basepairs in length can induce retinal degeneration in animal models(Kleinman et al. (2012) Mol. Ther. 20(1): 101-108).

The use of engineered nucleases for cleaving DNA targets in the humanRHO gene was previously disclosed in U.S. Patent Publication No. US2012/0204282 A1 by Zhang (“the Zhang application”). The Zhangapplication disclosed several approaches for targeting and modulatingthe expression of mutant RHO alleles. Specifically, the Zhangapplication discussed the use of engineered DNA binding domains, such aszinc finger proteins (ZFP) and TAL effector (TALE) proteins, asrepressors of RHO gene expression. The Zhang application also describedfusion proteins comprising a ZFP or TALE binding domain operably linkedto a regulatory or functional domain. The functional domain could be atranscriptional repressor domain that downregulates RHO gene expression.Alternatively, the functional domain could be a transcriptionalactivation domain. Further, the functional domain could comprise anuclease domain. When linked to a nuclease domain, the resulting fusionproteins include zinc finger nucleases (ZFNs) and TALE-nucleases(TALENs).

In addition to ZFNs and TALENs, the Zhang application discusses the useof meganucleases for targeting and inhibiting the expression ofwild-type and/or mutant RHO alleles. The Zhang application describes theuse of such meganucleases for disrupting RHO gene expression vianon-homologous end joining (NHEJ) at the recognition sequence, and forintroducing a replacement wild-type RHO gene sequence to express thewild-type rhodopsin protein. However, the recognition sequences in theRHO gene that are identified by the Zhang application are limited tothree pairs of ZFN target sites found in the wild-type RHO gene (see,Zhang application at Table 2).

The use of engineered meganucleases for cleaving DNA targets in the RHOgene was also disclosed in U.S. Patent Publication No. US 2013/0183282by Lemaire and Arnould (“the Lemaire application”). The Lemaireapplication disclosed meganucleases designed to target various regionsof the RHO gene for use in one of three gene therapy strategies. Thefirst strategy is gene correction, wherein the engineered meganucleasesare specific for a recognition sequence in the vicinity of a specifiedmutation, induce a double-strand break at that site, and rely onhomologous recombination of a corresponding non-mutant allelic sequenceinto the genome. The second strategy disclosed in the Lemaireapplication is exon knock-in, wherein a functional protein isreconstituted by using a meganuclease to introduce a synthetic wild-typecoding sequence into the genome while preventing the expression of thepathologic mutation. The third strategy disclosed in the Lemaireapplication is gene inactivation by mutagenesis, which relies ameganuclease to induce a double-strand break at a target recognitionsequence in the genome, and NHEJ at the cleavage site to induce amutation.

Accordingly, there is still a need in the art for methods that canpreferentially target and inactivate the RHO P23H allele for treatmentof RP.

SUMMARY OF THE INVENTION

The present invention provides recombinant meganucleases that areengineered to recognize and cleave P23H recognition sequences. Thepresent invention further provides methods comprising the delivery ofsuch a recombinant meganuclease, or genes encoding such a recombinantmeganuclease, to the cells of a patient having RP, such that therecombinant meganuclease (or encoded recombinant meganuclease)preferentially targets and cleaves a P23H recognition sequence presenton the mutant RHO P23H allele. NHEJ occurs at the cleavage site,resulting in mutagenesis and disruption of the mutant RHO P23H allele,while the functional wild-type RHO allele remains intact to expresswild-type rhodopsin in rod photoreceptor cells of the retina.Preferential inactivation of the mutant RHO P23H allele, and disruptionof P23H rhodopsin expression, is expected to delay, prevent, or reversethe progression of RP in patients.

Thus, in some embodiments, the present invention provides recombinantmeganucleases that are engineered to recognize and cleave a P23Hrecognition sequence, which is present in the mutant RHO P23H allele butnot in the wild-type RHO allele. The present invention further providesthe use of such a recombinant meganuclease in a method for treating RP,preferably autosomal dominant RP, wherein the mutant RHO P23H allele ispreferentially targeted and cleaved. In this manner, expression of P23Hrhodopsin is suppressed due to NHEJ at the meganuclease cleavage site,while the functional wild-type RHO allele remains intact to expresswild-type rhodopsin in rod photoreceptor cells of the retina.

Thus, the invention includes the use of site-specific, rare-cutting,homing endonucleases (also called “meganucleases”) that are engineeredto recognize specific DNA sequences in a locus of interest. Homingendonucleases are a group of naturally-occurring nucleases whichrecognize 15-40 base pair cleavage sites commonly found in the genomesof plants and fungi. They are frequently associated with parasitic DNAelements, such as group 1 self-splicing introns and inteins. Theynaturally promote homologous recombination or gene insertion at specificlocations in the host genome by producing a double-stranded break in thechromosome, which recruits the cellular DNA-repair machinery (Stoddard(2006), Q. Rev. Biophys. 38:49-95). Homing endonucleases are commonlygrouped into four families: the LAGLIDADG (SEQ ID NO:96) family, theGIY-YIG family, the His-Cys box family and the HNH family. Thesefamilies are characterized by structural motifs, which affect catalyticactivity and recognition sequence. For instance, members of theLAGLIDADG (SEQ ID NO:96) family are characterized by having either oneor two copies of the conserved LAGLIDADG (SEQ ID NO:96) motif (Chevalieret al. (2001), Nucleic Acids Res. 29(18): 3757-3774). The LAGLIDADG (SEQID NO:96) homing endonucleases with a single copy of the LAGLIDADG (SEQID NO:96) motif form homodimers, whereas members with two copies of theLAGLIDADG (SEQ ID NO:96) motif are found as monomers.

Methods for producing engineered, site-specific recombinantmeganucleases are known in the art. I-CreI (SEQ ID NO:95) is a member ofthe LAGLIDADG (SEQ ID NO:96) family of homing endonucleases whichrecognizes and cuts a 22 base pair recognition sequence in thechloroplast chromosome of the algae Chlamydomonas reinhardtii. Geneticselection techniques have been used to modify the wild-type I-CreIcleavage site preference (Sussman et al. (2004), J. Mol. Biol. 342:31-41; Chames et al. (2005), Nucleic Acids Res. 33: e178; Seligman etal. (2002), Nucleic Acids Res. 30: 3870-9, Arnould et al. (2006), J.Mol. Biol. 355: 443-58). More recently, a method of rationally-designingmono-LAGLIDADG (SEQ ID NO:96) homing endonucleases was described whichis capable of comprehensively redesigning I-CreI and other homingendonucleases to target widely-divergent DNA sites, including sites inmammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859).

As first described in WO 2009/059195, I-CreI and its engineeredderivatives are normally dimeric but can be fused into a singlepolypeptide using a short peptide linker that joins the C-terminus of afirst subunit to the N-terminus of a second subunit (see also Li, et al.(2009) Nucleic Acids Res. 37:1650-62; Grizot, et al. (2009) NucleicAcids Res. 37:5405-19.) Thus, a functional “single-chain” meganucleasecan be expressed from a single transcript. Such engineered meganucleasesexhibit an extremely low frequency of off-target cutting. By deliveringa gene encoding a single-chain meganuclease to retinal cells, andpreferably to rod photoreceptor cells, it is possible to specificallyand preferentially target, cleave, and inactivate the mutant RHO P23Hallele, thus suppressing expression of P23H rhodopsin.

Thus, in one aspect, the invention provides a recombinant meganucleasethat recognizes and cleaves a recognition sequence comprising themutation which encodes P23H substitution in the P23H mutant allele. Insome embodiments, the recognition sequence is selected from the SEQ IDNOs:1-4 (i.e., the P23H recognition sequences). The recombinantmeganuclease comprises a first subunit and a second subunit, wherein thefirst subunit recognizes a first recognition half-site of one of theP23H recognition sequences and comprises an (a) an amino acid sequencehaving at least 80% sequence identity to residues 198-344 of any one ofSEQ ID NOs:6-69 or residues 7-153 of any one of SEQ ID NOs:70-93; and(b) a first hypervariable (HVR1) region which determines specificity forthe first recognition half-site. The recombinant meganuclease furthercomprises a second subunit that recognizes a second half-site of one ofthe P23H recognition sequences and comprises: (a) an amino acid sequencehaving at least 80% sequence identity to residues 7-153 of any one ofSEQ ID NOs:6-69 or residues 198-344 of any one of SEQ ID NOs:70-93; and(b) a second hypervariable (HVR2) region which determines specificityfor the second recognition half-site. In some embodiments, the P23Hrecognition sequence is SEQ ID NO:1 and the recombinant meganucleasecomprises a first subunit and a second subunit, wherein the firstsubunit recognizes a first recognition half-site of one of SEQ ID NO:1and comprises (a) an amino acid sequence having at least 80% sequenceidentity to residues 198-344 of any one of SEQ ID NOs:6-69 or residues7-153 of any one of SEQ ID NOs:70-93; and (b) a first hypervariable(HVR1) region which determines specificity for the first recognitionhalf-site. The recombinant meganuclease further comprises a secondsubunit that recognizes a second half-site of SEQ ID NO:1 and comprises:(a) an amino acid sequence having at least 80% sequence identity toresidues 7-153 of any one of SEQ ID NOs:6-69 or residues 198-344 of anyone of SEQ ID NOs:70-93; and (b) a second hypervariable (HVR2) regionwhich determines specificity for the second recognition half-site.

In some embodiments, the P23H recognition sequence is SEQ ID NO:1 andthe first subunit comprises an amino acid sequence having at least 85%,90%, or 95% sequence identity to residues 198-344 of any one of SEQ IDNOs:6-69 or residues 7-153 of any one of SEQ ID NOs:70-93, and thesecond subunit comprises an amino acid sequence having at least 85%,90%, or 95% sequence identity to residues 7-153 of any one of SEQ IDNOs:6-69 or residues 198-344 of any one of SEQ ID NOs:70-93.

In other embodiments, the P23H recognition sequence is SEQ ID NO:1 andthe HVR1 region comprises W or Y at a position corresponding to: (a)position 215 of any one of SEQ ID NOs:6-69; or (b) position 24 of anyone of SEQ ID NOs:70-93. In other embodiments, the P23H recognitionsequence is SEQ ID NO:1 and the HVR1 region comprises I at a positioncorresponding to: (a) position 219 of any one of SEQ ID NOs:6-69; or (b)position 28 of any one of SEQ ID NOs:70-93. In other embodiments, theP23H recognition sequence is SEQ ID NO:1 and the HVR1 region comprises Vat a position corresponding to: (a) position 259 of any one of SEQ IDNOs:6-69; or (b) position 68 of any one of SEQ ID NOs:70-93. In otherembodiments, the P23H recognition sequence is SEQ ID NO:1 and the HVR1region comprises one or more of W or Y, I, and V at positionscorresponding to (a) positions 215, 219, and 259, respectively, of anyone of SEQ ID NOs:6-69; or (b) positions 24, 28, and 68, respectively,of any one of SEQ ID NOs:70-93.

In other embodiments, the P23H recognition sequence is SEQ ID NO:1 andthe HVR2 region comprises Y or M at a position corresponding to (a)position 24 of any one of SEQ ID NOs:6-69; or (b) position 215 of anyone of SEQ ID NOs:70-93. In other embodiments, the P23H recognitionsequence is SEQ ID NO:1 and the HVR2 region comprises F at a positioncorresponding to (a) position 28 of any one of SEQ ID NOs:6-69; or (b)position 219 of any one of SEQ ID NOs:70-93. In other embodiments, theP23H recognition sequence is SEQ ID NO:1 and the HVR2 region comprises Hat a position corresponding to (a) position 44 of any one of SEQ IDNOs:6-69; or (b) position 235 of any one of SEQ ID NOs:70-93. In otherembodiments, the P23H recognition sequence is SEQ ID NO:1 and the HVR2region comprises S at a position corresponding to (a) position 46 of anyone of SEQ ID NOs:6-69; or (b) position 237 of any one of SEQ IDNOs:70-93. In other embodiments, the P23H recognition sequence is SEQ IDNO:1 and the HVR2 region comprises W at a position corresponding to (a)position 70 of any one of SEQ ID NOs:6-69; or (b) position 261 of anyone of SEQ ID NOs:70-93. In other embodiments, the P23H recognitionsequence is SEQ ID NO:1 and the HVR2 region comprises one or more of Yor M, F, H, S, and W at positions corresponding to (a) positions 24, 28,44, 46, and 70, respectively, of any one of SEQ ID NOs:6-69; or (b)positions 215, 219, 235, 237, and 261, respectively, of any one of SEQID NOs:70-93.

In some embodiments, the recombinant meganuclease is a single-chainmeganuclease comprising a linker, wherein the linker covalently joinsthe first subunit and the second subunit.

In some embodiments, the P23H recognition sequence is SEQ ID NO:1 andthe first subunit comprises residues 198-344 of any one of SEQ IDNOs:6-69 or residues 7-153 of any one of SEQ ID NOs:70-93. In someembodiments, the P23H recognition sequence is SEQ ID NO:1 and the secondsubunit comprises residues 7-153 of any one of SEQ ID NOs:6-69 orresidues 198-344 of any one of SEQ ID NOs:70-93.

In some specific embodiments, the P23H recognition sequence is SEQ IDNO:1 and the meganuclease comprises the amino acid sequence of any oneof SEQ ID NOs:6-93.

In some embodiments, the recombinant meganuclease preferentiallyrecognizes and cleaves one of the P23H recognition sequences (SEQ IDNOs:1-4) relative to the corresponding 22 base pair recognition sequencepresent in the wild-type RHO allele (e.g., SE ID NO: 1 relative to SEQID NO:5).

In another aspect, the invention provides an isolated polynucleotidecomprising a nucleic acid sequence encoding a recombinant meganucleasedescribed herein.

In another aspect, the invention provides a recombinant DNA constructcomprising an isolated polynucleotide, wherein the isolatedpolynucleotide comprises a nucleic acid sequence encoding a recombinantmeganuclease described herein. In some embodiments, the recombinant DNAconstruct encodes a viral vector. In some such embodiments, the viralvector can be a retroviral vector, a lentiviral vector, an adenoviralvector, or an adeno-associated viral (AAV) vector. In some particularembodiment, the recombinant DNA construct encodes a recombinant AAVvector.

In another aspect, the invention provides a viral vector comprising anisolated polynucleotide, wherein the isolated polynucleotide comprises anucleic acid sequence encoding a recombinant meganuclease describedherein. In some embodiments, the viral vector can be a retroviralvector, a lentiviral vector, an adenoviral vector, or an AAV vector. Insome particular embodiments, the viral vector can be a recombinant AAVvector.

In another aspect, the invention provides a pharmaceutical compositionfor treatment of a subject having RP, preferably autosomal dominant RPcaused by the P23H mutation. The pharmaceutical composition can comprisea pharmaceutically acceptable carrier and: (a) a nucleic acid encoding arecombinant meganuclease described herein, wherein the recombinantmeganuclease is expressed in a target cell in vivo; or (b) a recombinantmeganuclease protein described herein; wherein the recombinantmeganuclease has specificity for one of the P23H recognition sequences(SEQ ID NOs:1-4) in the target cell.

In some embodiments, the nucleic acid encoding the recombinantmeganuclease can be an mRNA.

In other embodiments, the pharmaceutical composition comprises arecombinant DNA construct comprising the nucleic acid.

In some embodiments, the pharmaceutical composition comprises a viralvector comprising the nucleic acid. In one such embodiment, the viralvector can be a retroviral vector, a lentiviral vector, an adenoviralvector, or an AAV vector. In some particular embodiments, the viralvector can be a recombinant AAV vector.

In another aspect, the invention provides a recombinant meganucleasedescribed herein for use as a medicament. The invention further providesthe use of a recombinant meganuclease described herein in themanufacture of a medicament for treating RP, preferably autosomaldominant RP caused by the P23H mutation, and preferably for a subjectthat is heterozygous for the P23H mutant allele and a functional, normalor wild-type allele.

In another aspect, the invention provides an isolated polynucleotide foruse as a medicament, wherein the isolated polynucleotide comprises anucleic acid sequence encoding a recombinant meganuclease describedherein. The invention further provides the use of an isolatedpolynucleotide in the manufacture of a medicament for treating RP,preferably autosomal dominant RP caused by the P23H mutation, andpreferably for a subject that is heterozygous for the P23H mutant alleleand a functional, normal or wild-type allele, wherein the isolatedpolynucleotide comprises a nucleic acid sequence encoding a recombinantmeganuclease described herein.

In another aspect, the invention provides a recombinant AAV vector foruse as a medicament, wherein the recombinant AAV vector comprises anisolated polynucleotide, and wherein the isolated polynucleotidecomprises a nucleic acid sequence encoding a recombinant meganucleasedescribed herein. The invention further provides the use of arecombinant AAV vector in the manufacture of a medicament for treatingRP, preferably autosomal dominant RP caused by the P23H mutation, andpreferably for a subject that is heterozygous for the P23H mutant alleleand a functional, normal or wild-type allele wherein the recombinant AAVvector comprises an isolated polynucleotide, and wherein the isolatedpolynucleotide comprises a nucleic acid sequence encoding a recombinantmeganuclease described herein.

In another aspect, the invention provides a method for treating RPcaused by the P23H mutation in a subject in need thereof, and preferablyfor a subject that is heterozygous for the P23H mutant allele and afunctional, normal or wild-type allele. The method comprises contactingthe DNA of a target cell of said subject with a recombinant meganucleasedescribed herein that recognizes and cleaves the a recognition sequenceincluding the P23H mutation (e.g., SEQ ID NOs:1-4). The target cell ofthe subject comprises the P23H recognition sequence in a RHO geneallele, and cleavage of the recognition sequence inhibits expression ofthe RHO gene allele.

In some embodiments, the method is for treating autosomal dominant RPcaused by the P23H mutation, and preferably for a subject that isheterozygous for the P23H mutant allele and a functional, normal orwild-type allele.

In some embodiments, the target cell in the subject is a retinal targetcell. In preferred embodiments, the retinal cell is a rod photoreceptorcell.

In some embodiments of the method, a nucleic acid encoding therecombinant meganuclease is introduced into the target cell. In somesuch embodiments, the nucleic acid can be an mRNA. In other suchembodiments, the nucleic acid can be introduced into the cell using arecombinant DNA construct. In yet other such embodiments, the nucleicacid can be introduced into the target cell using a viral vector such asa retroviral vector, a lentiviral vector, an adenoviral vector, or anAAV vector. In some particular embodiment, a gene encoding therecombinant meganuclease is delivered to the target cell using arecombinant AAV vector.

In other embodiments of the method, a recombinant meganuclease proteinof the invention is introduced into the target cell.

In the various embodiments of the method, the recombinant meganucleaseprotein, or a nucleic acid encoding the recombinant meganuclease, can beadministered to the subject in a pharmaceutical composition describedherein.

In another aspect, the invention provides a method for producing agenetically-modified cell. The method comprises: (a) obtaining a cellcomprising at least one P23H RHO allele; and (b) introducing into thecell: (i) a nucleic acid sequence encoding a recombinant meganuclease ofthe invention, wherein the recombinant meganuclease is expressed in thecell; or (ii) a recombinant meganuclease protein; wherein therecombinant meganuclease has specificity for a P23H recognition sequencepresent on the P23H RHO allele(s); and wherein the recombinantmeganuclease recognizes and cleaves the P23H recognition sequence; andwherein expression of the P23H RHO allele is disrupted by non-homologousend joining at the cleavage site. Preferably the cell is heterozygousfor the P23H mutant allele and a functional, normal or wild-type alleleprior to the modification.

In some embodiments of the method, the cell can be a eukaryotic cell. Insome such embodiments, the eukaryotic cell can be a pluripotent cell. Insuch embodiments, the pluripotent cell can be an induced pluripotentstem (iPS) cell. In some particular embodiments, the iPS cell can be ahuman iPS cell.

In other embodiments of the method, the nucleic acid can be an mRNA.

In some embodiment of the method, the nucleic acid can be introducedinto the cell using a recombinant DNA construct.

In some embodiments of the method, the nucleic acid can be introducedinto the cell using a viral vector. In some such embodiments, the viralvector can be a retroviral vector, a lentiviral vector, an adenoviralvector, or an AAV vector. In some particular embodiments, the viralvector can be a recombinant AAV vector.

In another aspect, the invention provides a genetically-modified cell,wherein the genetically-modified cell comprises a wild-type RHO alleleand a disrupted P23H allele, wherein the genetically-modified cellexpresses a wild-type RHO protein and does not express a P23H RHOprotein, and wherein the genetically-modified cell is produced accordingto the methods of the invention described herein. In some embodiments,the disrupted P23H allele includes a deletion mutation caused bycleavage with the meganuclease and NHEJ. In some particular embodiments,the genetically-modified cell can be a pluripotent cell, an iPS cell, ora human iPS cell.

In another aspect, the invention provides a pharmaceutical compositionfor treatment of a subject having RP caused by the P23H mutation, andpreferably for a subject that is heterozygous for the P23H mutant alleleand a functional, normal or wild-type allele. In different embodiments,the pharmaceutical composition can comprise a pharmaceuticallyacceptable carrier and any genetically-modified cell of the invention,and/or any genetically-modified cell produced according to the methodsof the invention, which comprises a wild-type RHO allele that expresseswild-type RHO protein and a disrupted P23H allele which does not expressP23H RHO protein. In some embodiments, the disrupted P23H alleleincludes a deletion mutation caused by cleavage with the meganucleaseand NHEJ.

In another aspect, the invention provides a method for treating RPcaused by the P23H mutation in a subject in need thereof, and preferablyfor a subject that is heterozygous for the P23H mutant allele and afunctional, normal or wild-type allele. The method comprisesadministering to the subject a pharmaceutical composition describedherein which comprises a pharmaceutically acceptable carrier and agenetically-modified cell of the invention, which comprises a wild-typeRHO allele that expresses wild-type RHO protein and a disrupted P23Hallele which does not express P23H RHO protein. In some embodiments, thedisrupted P23H allele includes a deletion mutation caused by cleavagewith the meganuclease and NHEJ.

In some embodiments of the method, the genetically-modified cell can bedelivered to a target tissue. Such target tissues can include the eye,and particularly the retina.

Further, in some embodiments of the method, the genetically-modifiedcell can be a genetically-modified iPS cell. In such embodiments, thegenetically-modified iPS cell can differentiate into a cell whichexpresses wild-type RHO protein when it is delivered to the targettissue. In some particular embodiments, the genetically-modified iPScell can differentiate into a retinal cell, and particularly into a rodphotoreceptor cell, which expresses the wild-type rhodopsin protein butnot the P23H rhodopsin protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. P23H recognition sequence. A) Alignment of the 22 base pair P23Hrecognition sequence of SEQ ID NO:1 with the corresponding 22 base pairrecognition sequence present in the wild-type human RHO gene allele (SEQID NO:5). These sequences span nucleotides 49 to 70 of the P23H mutantor wild-type RHO gene coding sequences (SEQ ID NOs:98 and 97,respectively). The C68A nucleotide substitution within the P23Hrecognition sequence is highlighted. B) The P23H recognition sequence ofSEQ ID NO:1 comprises two recognition half-sites, referred to as RHO1and RHO2. Each recognition half-site comprises 9 base pairs as shown.Half-sites in the recognition sequence are separated by a 4 base paircentral region. C) The recombinant meganuclease of the inventioncomprises two subunits, wherein the first subunit binds to the a firsthalf-site (e.g., RHO1) of a P23H recognition sequence (e.g., SEQ IDNO:1) and the second subunit binds to a second half-site (e.g., RHO2).In embodiments where the recombinant meganuclease is a single-chainmeganuclease, the first subunit can be positioned as either theN-terminal or C-terminal subunit. Likewise, the second subunit can bepositioned as either the N-terminal or C-terminal subunit.

FIGS. 2A-2F. Amino acid alignment of RHO1-binding subunits. A-F)Recombinant meganucleases encompassed by the invention comprise onesubunit that binds the 9 base pair RHO1 recognition half-site of (SEQ IDNO:1. Amino acid sequence alignments are provided for the RHO1-bindingsubunits (SEQ ID NOs:102-188) of the recombinant meganucleases set forthin SEQ ID NOs:6-93. As shown, the RHO1-binding subunit of SEQ IDNOs:6-69 comprises residues 198-344, whereas the RHO1-binding subunit ofSEQ ID NOs:70-93 comprises residues 7-153. Each RHO1-binding subunitcomprises a 56 amino acid hypervariable region as indicated. Variableresidues within the hypervariable region are shaded, with the mostfrequent amino acids at each position further highlighted; the mostprevalent residues are bolded, whereas the second most prevalent arebolded and italicized. Residues outside of the hypervariable region areidentical in each subunit (but this is not required), with the exceptionof a Q or E residue at position 80 or position 271 (see, U.S. Pat. No.8,021,867). Nearly all RHO1-binding subunits provided in FIG. 2A-2Fshare at least 90% sequence identity to the RHO1-binding subunit(residues 198-344) of the RHO2-L3-59 meganuclease (SEQ ID NO:102).Residue numbers shown are those of SEQ ID NOs:6-93.

FIGS. 3A-3F. Amino acid alignment of RHO2-binding subunits. A-F)Recombinant meganucleases encompassed by the invention comprise onesubunit that binds the 9 base pair RHO2 recognition half-site of SEQ IDNO:1. Amino acid sequence alignments are provided for the RHO2-bindingsubunits (SEQ ID NOs:190-277) of the recombinant meganucleases set forthin SEQ ID NOs:6-93. As shown, the RHO2-binding subunit of SEQ IDNOs:6-69 comprises residues 7-153, whereas the RHO2-binding subunit ofSEQ ID NOs:70-93 comprises residues 198-344. Each RHO2-binding subunitcomprises a 56 amino acid hypervariable region as indicated. Variableresidues within the hypervariable region are shaded, with the mostfrequent amino acids at each position further highlighted; the mostprevalent residues are bolded, whereas the second most prevalent arebolded and italicized. Residues outside of the hypervariable region areidentical in each subunit (but this is not required), with the exceptionof a Q or E residue at position 80 or position 271 (see, U.S. Pat. No.8,021,867). Nearly all RHO2-binding subunits provided in FIGS. 3A-3Fshare at least 90% sequence identity to the RHO2-binding subunit(residues 7-153) of the RHO2-L3-59 meganuclease (SEQ ID NO:190). Residuenumbers shown are those of SEQ ID NOs:6-93.

FIG. 4. Schematic of reporter assay in CHO cells for evaluatingrecombinant meganucleases targeting a P23H recognition sequence. For therecombinant meganucleases described herein, a CHO cell line was producedin which a reporter cassette was integrated stably into the genome ofthe cell. The reporter cassette comprised, in 5′ to 3′ order: an SV40Early Promoter; the 5′ 2/3 of the GFP gene; the recognition sequence foran engineered meganuclease of the invention, for example a P23Hrecognition sequence of any one of SEQ ID NOs:1-4; the recognitionsequence for the CHO-23/24 meganuclease (WO 2012/167192); and the 3′ 2/3of the GFP gene. Cells stably transfected with this cassette did notexpress GFP in the absence of a DNA break-inducing agent. Meganucleaseswere introduced by transduction of plasmid DNA or mRNA encoding eachmeganuclease. When a DNA break was induced at either of the meganucleaserecognition sequences, the duplicated regions of the GFP gene recombinedwith one another to produce a functional GFP gene. The percentage ofGFP-expressing cells could then be determined by flow cytometry as anindirect measure of the frequency of genome cleavage by themeganucleases.

FIG. 5. Efficiency of recombinant meganucleases for recognizing andcleaving a P23H recognition sequence in a CHO cell reporter assay. A)-J)Each of the recombinant meganucleases set forth in SEQ ID NOs:6-93 wereengineered to target a P23H recognition sequence and were screened forefficacy in the CHO cell reporter assay. The results shown provide thepercentage of GFP-expressing cells observed in each assay, whichindicates the efficacy of each meganuclease for cleaving the P23Hrecognition sequence or the CHO-23/24 recognition sequence. A negativecontrol (RHO 1-2 bs) was further included in each assay.

FIG. 6. Time course of recombinant meganuclease efficacy in CHO cellreporter assay. Recombinant meganucleases encompassed by the inventionwere evaluated in the CHO reporter assay, with the percentage ofGFP-expressing cells determined 1, 4, 6, and 8 days after introductionof meganuclease-encoding mRNA into the CHO reporter cells. A CHO-23/24meganuclease was included at each time point as a positive control.

FIG. 7. Selectivity of recombinant meganucleases. A)-F) The selectivityof recombinant meganucleases for a P23H recognition sequence (SEQ IDNO:1) versus the corresponding wild-type RHO recognition sequence (SEQID NO:5) was determined using the CHO cell reporter assay. Recombinantmeganucleases encompassed by the invention were introduced into cellscomprising a P23H recognition sequence (“RHO 1-2 cells,” gray bars) orthe corresponding wild-type RHO recognition sequence (“RHO 3-4 cells,”black bars) to determine if they could discriminate between the mutantand wild-type targets.

FIG. 8. Generation and expression of recombinant AAV vectors. A) Diagramof recombinant AAV vector genome shown with inverted terminal repeats(ITRs) at the 5′ and 3′ ends. The vector comprises the coding sequencefor the recombinant meganuclease RHO-1/2-L2-49 (SEQ ID NO:8) operablylinked to a cytomegalovirus-early (CMV) promoter. The nucleaseexpression cassette was incorporated into a “packaging” plasmid that wasused in conjunction with an Ad helper plasmid to produce recombinant AAVcapable of delivering genes encoding the RHO-1/2-L2-49 meganuclease. B)Immunoblot of RHO-1/2-L2-49 meganuclease expression in recombinantAAV-transduced CHO cells after 24 hours.

FIG. 9. Efficiency and selectivity of a RHO-1/2-L2-49 meganuclease for aP23H recognition sequence (SEQ ID NO:1) when expressed by recombinantAAV in a CHO cell reporter assay. CHO cells harboring the wild-typerecognition sequence (SEQ ID NO:5; “RHO 3-4 cells”, black bars) or theP23H recognition sequence (SEQ ID NO:1; “RHO 1-2 cells,” gray bars) weretransduced with three different doses of AAV2 vector encoding theRHO-1/2-L2-49 meganuclease operably linked to a CMV promoter.

FIG. 10. RHO-1/2-L2-49 meganuclease stability in CHO reporter cellsfollowing treatment with cycloheximide.

FIG. 11. A) Vector map of the pDS CMV RHO2_L3_59 plasmid (SEQ IDNO:278). B) Vector map of the pDS CMV RHO2_L5_14 plasmid (SEQ IDNO:279).

FIG. 12. CHO GFFP reporter assay demonstrating specificity of RHO 1-2meganucleases for RHO 1-2 recognition sequence. CHO-K cells (controls),WT RHO cells (comprising wild-type RHO sequence of SEQ ID NO:5), or P23HRHO cells (comprising P23H RHO sequence of SEQ ID NO:1) were transducedwith low, medium, or high titers of recombinant AAV vectors encoding aGFP protein, the RHO2-L3-59 meganuclease (SEQ ID NO:6), or theRHO2-L5-14 meganuclease (SEQ ID NO:7). The percent of GFP-positive cellswas determined in each cell line following transduction as a measure ofrecognition sequence cleavage.

FIG. 13. Western blot analysis of meganuclease expression in CHO RHO 1-2cells following transduction of recombinant AAV vectors. P23H RHO cells,comprising the RHO 1-2 recognition sequence, were transduced with 1e8,1e9, or 2e9 viral genomes per cell of recombinant AAV vectors encodingthe RHO2-L3-59 meganuclease (SEQ ID NO:6) or the RHO2-L5-14 meganuclease(SEQ ID NO:7). Cell lysates were analyzed for expression of GFP as ameasure of RHO 1-2 recognition sequence cleavage (top panel),meganuclease expression (bottom panel), and β-actin as a loadingcontrol. Lanes 1-6 were transduced to express the RHO2-L3-59meganuclease. Lanes 7-12 were transduced to express the RHO2-L5-14meganuclease.

FIG. 14. Vector map of the pDS GRK1 RHO2_L5_14 plasmid (SEQ ID NO:281).

FIG. 15. Western blot analysis of meganuclease expression in mouseretinal cells following subretinal AAV injection. Wild-type mice wereadministered a recombinant AAV vector encoding the RHO2-L5-14meganuclease (SEQ ID NO:7) by subretinal injection. Retinal cells wereobtained from the left (OS) and right (OD) eye of five mice and celllysates were analyzed by Western blot for the expression of theRHO2-L5-14 meganuclease and β-actin.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 sets forth the nucleotide sequence of one P23H recognitionsequence (RHO-1/2).

SEQ ID NO:2 sets forth the nucleotide sequence of one P23H recognitionsequence (RHO-9/10).

SEQ ID NO:3 sets forth the nucleotide sequence of one P23H recognitionsequence (RHO-11/12).

SEQ ID NO:4 sets forth the nucleotide sequence of one P23H recognitionsequence (RHO-13/14).

SEQ ID NO:5 sets forth the nucleotide sequence of the correspondingrecognition sequence found in the wild-type RHO allele (i.e., the RHO3-4 recognition sequence).

SEQ ID NO: 6 sets forth the amino acid sequence of the RHO2-L3-59meganuclease.

SEQ ID NO: 7 sets forth the amino acid sequence of the RHO2-L5-14meganuclease.

SEQ ID NO: 8 sets forth the amino acid sequence of the RHO-1/2-L2-49meganuclease.

SEQ ID NO: 9 sets forth the amino acid sequence of the RHO 1-2x.179meganuclease.

SEQ ID NO: 10 sets forth the amino acid sequence of the RHO 1-2x.4meganuclease.

SEQ ID NO: 11 sets forth the amino acid sequence of the RHO 1-2x.207meganuclease.

SEQ ID NO: 12 sets forth the amino acid sequence of the RHO 1-2x.277meganuclease.

SEQ ID NO: 13 sets forth the amino acid sequence of the RHO 1-2x.292meganuclease.

SEQ ID NO: 14 sets forth the amino acid sequence of the RHO 1-2x.324meganuclease.

SEQ ID NO: 15 sets forth the amino acid sequence of the RHO 1-2x.371meganuclease.

SEQ ID NO: 16 sets forth the amino acid sequence of the RHO 1-2x.164meganuclease.

SEQ ID NO: 17 sets forth the amino acid sequence of the RHO 1-2x.181meganuclease.

SEQ ID NO: 18 sets forth the amino acid sequence of the RHO 1-2x.184meganuclease.

SEQ ID NO: 19 sets forth the amino acid sequence of the RHO-1/2-L1-21meganuclease.

SEQ ID NO: 20 sets forth the amino acid sequence of the RHO-1/2-L1-43meganuclease.

SEQ ID NO: 21 sets forth the amino acid sequence of the RHO-1/2-L1-45meganuclease.

SEQ ID NO: 22 sets forth the amino acid sequence of the RHO-1/2-L1-60meganuclease.

SEQ ID NO: 23 sets forth the amino acid sequence of the RHO-1/2-L1-61meganuclease.

SEQ ID NO: 24 sets forth the amino acid sequence of the RHO-1/2-L1-58meganuclease.

SEQ ID NO: 25 sets forth the amino acid sequence of the RHO-1/2-L1-7meganuclease.

SEQ ID NO: 26 sets forth the amino acid sequence of the RHO-1/2-L1-13meganuclease.

SEQ ID NO: 27 sets forth the amino acid sequence of the RHO-1/2-L1-18meganuclease.

SEQ ID NO: 28 sets forth the amino acid sequence of the RHO-1/2-L1-70meganuclease.

SEQ ID NO: 29 sets forth the amino acid sequence of the RHO-1/2-L1-86meganuclease.

SEQ ID NO: 30 sets forth the amino acid sequence of the RHO-1/2-L2-13meganuclease.

SEQ ID NO: 31 sets forth the amino acid sequence of the RHO-1/2-L2-24meganuclease.

SEQ ID NO: 32 sets forth the amino acid sequence of the RHO-1/2-L2-37meganuclease.

SEQ ID NO: 33 sets forth the amino acid sequence of the RHO-1/2-L2-58meganuclease.

SEQ ID NO: 34 sets forth the amino acid sequence of the RHO-1/2-L2-31meganuclease.

SEQ ID NO: 35 sets forth the amino acid sequence of the RHO-1/2-L2-29meganuclease.

SEQ ID NO: 36 sets forth the amino acid sequence of the RHO-1/2-L2-61meganuclease.

SEQ ID NO: 37 sets forth the amino acid sequence of the RHO2-L3-2meganuclease.

SEQ ID NO: 38 sets forth the amino acid sequence of the RHO2-L3-3meganuclease.

SEQ ID NO: 39 sets forth the amino acid sequence of the RI-102-L3-5meganuclease.

SEQ ID NO: 40 sets forth the amino acid sequence of the RHO2-L3-10meganuclease.

SEQ ID NO: 41 sets forth the amino acid sequence of the RHO2-L3-11meganuclease.

SEQ ID NO: 42 sets forth the amino acid sequence of the RHO2-L3-12meganuclease.

SEQ ID NO: 43 sets forth the amino acid sequence of the RHO2-L3-13meganuclease.

SEQ ID NO: 44 sets forth the amino acid sequence of the RHO2-L3-28meganuclease.

SEQ ID NO: 45 sets forth the amino acid sequence of the RHO2-L3-29meganuclease.

SEQ ID NO: 46 sets forth the amino acid sequence of the RHO2-L3-57meganuclease.

SEQ ID NO: 47 sets forth the amino acid sequence of the RHO2-L3-80meganuclease.

SEQ ID NO: 48 sets forth the amino acid sequence of the RHO2-L3-85meganuclease.

SEQ ID NO: 49 sets forth the amino acid sequence of the RHO2-L3-86meganuclease.

SEQ ID NO: 50 sets forth the amino acid sequence of the RHO2-L3-92meganuclease.

SEQ ID NO: 51 sets forth the amino acid sequence of the RHO2-L3-4meganuclease.

SEQ ID NO: 52 sets forth the amino acid sequence of the RHO2-L3-20meganuclease.

SEQ ID NO: 53 sets forth the amino acid sequence of the RHO2-L3-72meganuclease.

SEQ ID NO: 54 sets forth the amino acid sequence of the RHO1-L1-4meganuclease.

SEQ ID NO: 55 sets forth the amino acid sequence of the RHO1-L1-8meganuclease.

SEQ ID NO: 56 sets forth the amino acid sequence of the RHO1-L1-13meganuclease.

SEQ ID NO: 57 sets forth the amino acid sequence of the RHO1-L1-19meganuclease.

SEQ ID NO: 58 sets forth the amino acid sequence of the RHO1-L1-58meganuclease.

SEQ ID NO: 59 sets forth the amino acid sequence of the RHO1-L1-69meganuclease.

SEQ ID NO: 60 sets forth the amino acid sequence of the RHO1-L1-80meganuclease.

SEQ ID NO: 61 sets forth the amino acid sequence of the RHO1-L1-82meganuclease.

SEQ ID NO: 62 sets forth the amino acid sequence of the RHO1-L1-73meganuclease.

SEQ ID NO: 63 sets forth the amino acid sequence of the RHO1-L1-85meganuclease.

SEQ ID NO: 64 sets forth the amino acid sequence of the RHO1-L1-86meganuclease.

SEQ ID NO: 65 sets forth the amino acid sequence of the RHO-1/2-L4-10meganuclease.

SEQ ID NO: 66 sets forth the amino acid sequence of the RHO-1/2-L4-29meganuclease.

SEQ ID NO: 67 sets forth the amino acid sequence of the RHO-1/2-L4-65meganuclease.

SEQ ID NO: 68 sets forth the amino acid sequence of the RHO-1/2-L4-66meganuclease.

SEQ ID NO: 69 sets forth the amino acid sequence of the RHO-1/2-L4-85meganuclease.

SEQ ID NO: 70 sets forth the amino acid sequence of the RHO 1-2x.216meganuclease.

SEQ ID NO: 71 sets forth the amino acid sequence of the RHO 1-2x.241meganuclease.

SEQ ID NO: 72 sets forth the amino acid sequence of the RHO 1-2x.94meganuclease.

SEQ ID NO: 73 sets forth the amino acid sequence of the RHO 1-2x.95meganuclease.

SEQ ID NO: 74 sets forth the amino acid sequence of the RHO 1-2x.1meganuclease.

SEQ ID NO: 75 sets forth the amino acid sequence of the RHO 1-2x.60meganuclease.

SEQ ID NO: 76 sets forth the amino acid sequence of the RHO 1-2x.74meganuclease.

SEQ ID NO: 77 sets forth the amino acid sequence of the RHO 1-2x.88meganuclease.

SEQ ID NO: 78 sets forth the amino acid sequence of the RHO 1-2x.294meganuclease.

SEQ ID NO: 79 sets forth the amino acid sequence of the RHO 1-2x.302meganuclease.

SEQ ID NO: 80 sets forth the amino acid sequence of the RHO 1-2x.306meganuclease.

SEQ ID NO: 81 sets forth the amino acid sequence of the RHO 1-2x.338meganuclease.

SEQ ID NO: 82 sets forth the amino acid sequence of the RHO 1-2x.348meganuclease.

SEQ ID NO: 83 sets forth the amino acid sequence of the RHO 1-2x.356meganuclease.

SEQ ID NO: 84 sets forth the amino acid sequence of the RHO 1-2x.364meganuclease.

SEQ ID NO: 85 sets forth the amino acid sequence of the RHO 1-2x.142meganuclease.

SEQ ID NO: 86 sets forth the amino acid sequence of the RHO 1-2x.177meganuclease.

SEQ ID NO: 87 sets forth the amino acid sequence of the RHO 1-2x.148meganuclease.

SEQ ID NO: 88 sets forth the amino acid sequence of the RHO 1-2x.20meganuclease.

SEQ ID NO: 89 sets forth the amino acid sequence of the RHO 1-2x.55meganuclease.

SEQ ID NO: 90 sets forth the amino acid sequence of the RHO 1-2x.197meganuclease.

SEQ ID NO: 91 sets forth the amino acid sequence of the RHO 1-2x.252meganuclease.

SEQ ID NO: 92 sets forth the amino acid sequence of the RHO 1-2x.372meganuclease.

SEQ ID NO: 93 sets forth the amino acid sequence of the RHO 1-2x.151meganuclease.

SEQ ID NO:94 sets forth the amino acid sequence of the wild-type I-CreImeganuclease.

SEQ ID NO:95 sets forth the amino acid sequence of the LAGLIDADG motifof the I-CreI meganuclease.

SEQ ID NO:96 sets forth the nucleic acid sequence of the coding regionfor wild-type human rhodopsin.

SEQ ID NO:97 sets forth the nucleic acid sequence of the coding regionfor mutant P23H rhodopsin.

SEQ ID NO:98 sets forth the nucleic acid sequence of the wild-type humanrhodopsin gene.

SEQ ID NO:99 sets forth the nucleic acid sequence of the human rhodopsingene comprising a C68A mutation that encodes a P23H substitution inrhodopsin.

SEQ ID NO:100 sets forth the amino acid sequence of wild-type humanrhodopsin.

SEQ ID NO:101 sets for the amino acid sequence of mutant P23H rhodopsin.

SEQ ID NO: 102 sets forth residues 198-344 of the RHO2-L3-59meganuclease.

SEQ ID NO: 103 sets forth residues 198-344 of the RHO2-L5-14meganuclease.

SEQ ID NO: 104 sets forth residues 198-344 of the RHO-1/2-L2-49meganuclease.

SEQ ID NO: 105 sets forth residues 198-344 of the RHO 1-2x.179meganuclease.

SEQ ID NO: 106 sets forth residues 198-344 of the RHO 1-2x.4meganuclease.

SEQ ID NO: 107 sets forth residues 198-344 of the RHO 1-2x.207meganuclease.

SEQ ID NO: 108 sets forth residues 198-344 of the RHO 1-2x.277meganuclease.

SEQ ID NO: 109 sets forth residues 198-344 of the RHO 1-2x.292meganuclease.

SEQ ID NO: 110 sets forth residues 198-344 of the RHO 1-2x.324meganuclease.

SEQ ID NO: 111 sets forth residues 198-344 of the RHO 1-2x.371meganuclease.

SEQ ID NO: 112 sets forth residues 198-344 of the RHO 1-2x.164meganuclease.

SEQ ID NO: 113 sets forth residues 198-344 of the RHO 1-2x.181meganuclease.

SEQ ID NO: 114 sets forth residues 198-344 of the RHO 1-2x.184meganuclease.

SEQ ID NO: 115 sets forth residues 198-344 of the RHO-1/2-L1-21meganuclease.

SEQ ID NO: 116 sets forth residues 198-344 of the RHO-1/2-L1-43meganuclease.

SEQ ID NO: 117 sets forth residues 198-344 of the RHO-1/2-L1-45meganuclease.

SEQ ID NO: 118 sets forth residues 198-344 of the RHO-1/2-L1-60meganuclease.

SEQ ID NO: 119 sets forth residues 198-344 of the RHO-1/2-L1-61meganuclease.

SEQ ID NO: 120 sets forth residues 198-344 of the RHO-1/2-L1-58meganuclease.

SEQ ID NO: 121 sets forth residues 198-344 of the RHO-1/2-L1-7meganuclease.

SEQ ID NO: 122 sets forth residues 198-344 of the RHO-1/2-L1-13meganuclease.

SEQ ID NO: 123 sets forth residues 198-344 of the RHO-1/2-L1-18meganuclease.

SEQ ID NO: 124 sets forth residues 198-344 of the RHO-1/2-L1-70meganuclease.

SEQ ID NO: 125 sets forth residues 198-344 of the RHO-1/2-L1-86meganuclease.

SEQ ID NO: 126 sets forth residues 198-344 of the RHO-1/2-L2-13meganuclease.

SEQ ID NO: 127 sets forth residues 198-344 of the RHO-1/2-L2-24meganuclease.

SEQ ID NO: 128 sets forth residues 198-344 of the RHO-1/2-L2-37meganuclease.

SEQ ID NO: 129 sets forth residues 198-344 of the RHO-1/2-L2-58meganuclease.

SEQ ID NO: 130 sets forth residues 198-344 of the RHO-1/2-L2-31meganuclease.

SEQ ID NO: 131 sets forth residues 198-344 of the RHO-1/2-L2-29meganuclease.

SEQ ID NO: 132 sets forth residues 198-344 of the RHO-1/2-L2-61meganuclease.

SEQ ID NO: 133 sets forth residues 198-344 of the RHO2-L3-2meganuclease.

SEQ ID NO: 134 sets forth residues 198-344 of the RHO2-L3-3meganuclease.

SEQ ID NO: 135 sets forth residues 198-344 of the RHO2-L3-5meganuclease.

SEQ ID NO: 136 sets forth residues 198-344 of the RHO2-L3-10meganuclease.

SEQ ID NO: 137 sets forth residues 198-344 of the RHO2-L3-11meganuclease.

SEQ ID NO: 138 sets forth residues 198-344 of the RHO2-L3-12meganuclease.

SEQ ID NO: 139 sets forth residues 198-344 of the RHO2-L3-13meganuclease.

SEQ ID NO: 140 sets forth residues 198-344 of the RHO2-L3-28meganuclease.

SEQ ID NO: 141 sets forth residues 198-344 of the RHO2-L3-29meganuclease.

SEQ ID NO: 142 sets forth residues 198-344 of the RHO2-L3-57meganuclease.

SEQ ID NO: 143 sets forth residues 198-344 of the RHO2-L3-80meganuclease.

SEQ ID NO: 144 sets forth residues 198-344 of the RHO2-L3-85meganuclease.

SEQ ID NO: 145 sets forth residues 198-344 of the RHO2-L3-86meganuclease.

SEQ ID NO: 146 sets forth residues 198-344 of the RHO2-L3-92meganuclease.

SEQ ID NO: 147 sets forth residues 198-344 of the RHO2-L3-4meganuclease.

SEQ ID NO: 148 sets forth residues 198-344 of the RHO2-L3-20meganuclease.

SEQ ID NO: 149 sets forth residues 198-344 of the RHO2-L3-72meganuclease.

SEQ ID NO: 150 sets forth residues 198-344 of the RHO1-L1-4meganuclease.

SEQ ID NO: 151 sets forth residues 198-344 of the RHO1-L1-8meganuclease.

SEQ ID NO: 152 sets forth residues 198-344 of the RHO1-L1-13meganuclease.

SEQ ID NO: 153 sets forth residues 198-344 of the RHO1-L1-19meganuclease.

SEQ ID NO: 154 sets forth residues 198-344 of the RHO1-L1-58meganuclease.

SEQ ID NO: 155 sets forth residues 198-344 of the RHO1-L1-69meganuclease.

SEQ ID NO: 156 sets forth residues 198-344 of the RHO1-L1-80meganuclease.

SEQ ID NO: 157 sets forth residues 198-344 of the RHO1-L1-82meganuclease.

SEQ ID NO: 158 sets forth residues 198-344 of the RHO1-L1-73meganuclease.

SEQ ID NO: 159 sets forth residues 198-344 of the RHO1-L1-85meganuclease.

SEQ ID NO: 160 sets forth residues 198-344 of the RHO1-L1-86meganuclease.

SEQ ID NO: 161 sets forth residues 198-344 of the RHO-1/2-L4-10meganuclease.

SEQ ID NO: 162 sets forth residues 198-344 of the RHO-1/2-L4-29meganuclease.

SEQ ID NO: 163 sets forth residues 198-344 of the RHO-1/2-L4-65meganuclease.

SEQ ID NO: 164 sets forth residues 198-344 of the RHO-1/2-L4-66meganuclease.

SEQ ID NO: 165 sets forth residues 198-344 of the RHO-1/2-L4-85meganuclease.

SEQ ID NO: 166 sets forth residues 7-153 of the RHO 1-2x.216meganuclease.

SEQ ID NO: 167 sets forth residues 7-153 of the RHO 1-2x.241meganuclease.

SEQ ID NO: 168 sets forth residues 7-153 of the RHO 1-2x.94meganuclease.

SEQ ID NO: 169 sets forth residues 7-153 of the RHO 1-2x.95meganuclease.

SEQ ID NO: 170 sets forth residues 7-153 of the RHO 1-2x.1 meganuclease.

SEQ ID NO: 171 sets forth residues 7-153 of the RHO 1-2x.60meganuclease.

SEQ ID NO: 172 sets forth residues 7-153 of the RHO 1-2x.74meganuclease.

SEQ ID NO: 173 sets forth residues 7-153 of the RHO 1-2x.88meganuclease.

SEQ ID NO: 174 sets forth residues 7-153 of the RHO 1-2x.294meganuclease.

SEQ ID NO: 175 sets forth residues 7-153 of the RHO 1-2x.302meganuclease.

SEQ ID NO: 176 sets forth residues 7-153 of the RHO 1-2x.306meganuclease.

SEQ ID NO: 177 sets forth residues 7-153 of the RHO 1-2x.338meganuclease.

SEQ ID NO: 178 sets forth residues 7-153 of the RHO 1-2x.348meganuclease.

SEQ ID NO: 179 sets forth residues 7-153 of the RHO 1-2x.356meganuclease.

SEQ ID NO: 180 sets forth residues 7-153 of the RHO 1-2x.364meganuclease.

SEQ ID NO: 181 sets forth residues 7-153 of the RHO 1-2x.142meganuclease.

SEQ ID NO: 182 sets forth residues 7-153 of the RHO 1-2x.177meganuclease.

SEQ ID NO: 183 sets forth residues 7-153 of the RHO 1-2x.148meganuclease.

SEQ ID NO: 184 sets forth residues 7-153 of the RHO 1-2x.20meganuclease.

SEQ ID NO: 185 sets forth residues 7-153 of the RHO 1-2x.55meganuclease.

SEQ ID NO: 186 sets forth residues 7-153 of the RHO 1-2x.197meganuclease.

SEQ ID NO: 187 sets forth residues 7-153 of the RHO 1-2x.252meganuclease.

SEQ ID NO: 188 sets forth residues 7-153 of the RHO 1-2x.372meganuclease.

SEQ ID NO: 189 sets forth residues 7-153 of the RHO 1-2x.151meganuclease.

SEQ ID NO: 190 sets forth residues 7-153 of the RHO2-L3-59 meganuclease.

SEQ ID NO: 191 sets forth residues 7-153 of the RHO2-L5-14 meganuclease.

SEQ ID NO: 192 sets forth residues 7-153 of the RHO-1/2-L2-49meganuclease.

SEQ ID NO: 193 sets forth residues 7-153 of the RHO 1-2x.179meganuclease.

SEQ ID NO: 194 sets forth residues 7-153 of the RHO 1-2x.4 meganuclease.

SEQ ID NO: 195 sets forth residues 7-153 of the RHO 1-2x.207meganuclease.

SEQ ID NO: 196 sets forth residues 7-153 of the RHO 1-2x.277meganuclease.

SEQ ID NO: 197 sets forth residues 7-153 of the RHO 1-2x.292meganuclease.

SEQ ID NO: 198 sets forth residues 7-153 of the RHO 1-2x.324meganuclease.

SEQ ID NO: 199 sets forth residues 7-153 of the RHO 1-2x.371meganuclease.

SEQ ID NO: 200 sets forth residues 7-153 of the RHO 1-2x.164meganuclease.

SEQ ID NO: 201 sets forth residues 7-153 of the RHO 1-2x.181meganuclease.

SEQ ID NO: 202 sets forth residues 7-153 of the RHO 1-2x.184meganuclease.

SEQ ID NO: 203 sets forth residues 7-153 of the RHO-1/2-L1-21meganuclease.

SEQ ID NO: 204 sets forth residues 7-153 of the RHO-1/2-L1-43meganuclease.

SEQ ID NO: 205 sets forth residues 7-153 of the RHO-1/2-L1-45meganuclease.

SEQ ID NO: 206 sets forth residues 7-153 of the RHO-1/2-L1-60meganuclease.

SEQ ID NO: 207 sets forth residues 7-153 of the RHO-1/2-L1-61meganuclease.

SEQ ID NO: 208 sets forth residues 7-153 of the RHO-1/2-L1-58meganuclease.

SEQ ID NO: 209 sets forth residues 7-153 of the RHO-1/2-L1-7meganuclease.

SEQ ID NO: 210 sets forth residues 7-153 of the RHO-1/2-L1-13meganuclease.

SEQ ID NO: 211 sets forth residues 7-153 of the RHO-1/2-L1-18meganuclease.

SEQ ID NO: 212 sets forth residues 7-153 of the RHO-1/2-L1-70meganuclease.

SEQ ID NO: 213 sets forth residues 7-153 of the RHO-1/2-L1-86meganuclease.

SEQ ID NO: 214 sets forth residues 7-153 of the RHO-1/2-L2-13meganuclease.

SEQ ID NO: 215 sets forth residues 7-153 of the RHO-1/2-L2-24meganuclease.

SEQ ID NO: 216 sets forth residues 7-153 of the RHO-1/2-L2-37meganuclease.

SEQ ID NO: 217 sets forth residues 7-153 of the RHO-1/2-L2-58meganuclease.

SEQ ID NO: 218 sets forth residues 7-153 of the RHO-1/2-L2-31meganuclease.

SEQ ID NO: 219 sets forth residues 7-153 of the RHO-1/2-L2-29meganuclease.

SEQ ID NO: 220 sets forth residues 7-153 of the RHO-1/2-L2-61meganuclease.

SEQ ID NO: 221 sets forth residues 7-153 of the RHO2-L3-2 meganuclease.

SEQ ID NO: 222 sets forth residues 7-153 of the RHO2-L3-3 meganuclease.

SEQ ID NO: 223 sets forth residues 7-153 of the RHO2-L3-5 meganuclease.

SEQ ID NO: 224 sets forth residues 7-153 of the RHO2-L3-10 meganuclease.

SEQ ID NO: 225 sets forth residues 7-153 of the RHO2-L3-11 meganuclease.

SEQ ID NO: 226 sets forth residues 7-153 of the RHO2-L3-12 meganuclease.

SEQ ID NO: 227 sets forth residues 7-153 of the RHO2-L3-13 meganuclease.

SEQ ID NO: 228 sets forth residues 7-153 of the RHO2-L3-28 meganuclease.

SEQ ID NO: 229 sets forth residues 7-153 of the RHO2-L3-29 meganuclease.

SEQ ID NO: 230 sets forth residues 7-153 of the RHO2-L3-57 meganuclease.

SEQ ID NO: 231 sets forth residues 7-153 of the RHO2-L3-80 meganuclease.

SEQ ID NO: 232 sets forth residues 7-153 of the RHO2-L3-85 meganuclease.

SEQ ID NO: 233 sets forth residues 7-153 of the RHO2-L3-86 meganuclease.

SEQ ID NO: 234 sets forth residues 7-153 of the RHO2-L3-92 meganuclease.

SEQ ID NO: 235 sets forth residues 7-153 of the RHO2-L3-4 meganuclease.

SEQ ID NO: 236 sets forth residues 7-153 of the RHO2-L3-20 meganuclease.

SEQ ID NO: 237 sets forth residues 7-153 of the RHO2-L3-72 meganuclease.

SEQ ID NO: 238 sets forth residues 7-153 of the RHO1-L1-4 meganuclease.

SEQ ID NO: 239 sets forth residues 7-153 of the RHO1-L1-8 meganuclease.

SEQ ID NO: 240 sets forth residues 7-153 of the RHO1-L1-13 meganuclease.

SEQ ID NO: 241 sets forth residues 7-153 of the RHO1-L1-19 meganuclease.

SEQ ID NO: 242 sets forth residues 7-153 of the RHO1-L1-58 meganuclease.

SEQ ID NO: 243 sets forth residues 7-153 of the RHO1-L1-69 meganuclease.

SEQ ID NO: 244 sets forth residues 7-153 of the RHO1-L1-80 meganuclease.

SEQ ID NO: 245 sets forth residues 7-153 of the RHO1-L1-82 meganuclease.

SEQ ID NO: 246 sets forth residues 7-153 of the RHO1-L1-73 meganuclease.

SEQ ID NO: 244 sets forth residues 7-153 of the RHO1-L1-85 meganuclease.

SEQ ID NO: 248 sets forth residues 7-153 of the RHO1-L1-86 meganuclease.

SEQ ID NO: 249 sets forth residues 7-153 of the RHO-1/2-L4-10meganuclease.

SEQ ID NO: 250 sets forth residues 7-153 of the RHO-1/2-L4-29meganuclease.

SEQ ID NO: 251 sets forth residues 7-153 of the RHO-1/2-L4-65meganuclease.

SEQ ID NO: 252 sets forth residues 7-153 of the RHO-1/2-L4-66meganuclease.

SEQ ID NO: 253 sets forth residues 7-153 of the RHO-1/2-L4-85meganuclease.

SEQ ID NO: 254 sets forth residues 198-344 of the RHO 1-2x.216meganuclease.

SEQ ID NO: 255 sets forth residues 198-344 of the RHO 1-2x.241meganuclease.

SEQ ID NO: 256 sets forth residues 198-344 of the RHO 1-2x.94meganuclease.

SEQ ID NO: 257 sets forth residues 198-344 of the RHO 1-2x.95meganuclease.

SEQ ID NO: 258 sets forth residues 198-344 of the RHO 1-2x.1meganuclease.

SEQ ID NO: 259 sets forth residues 198-344 of the RHO 1-2x.60meganuclease.

SEQ ID NO: 260 sets forth residues 198-344 of the RHO 1-2x.74meganuclease.

SEQ ID NO: 261 sets forth residues 198-344 of the RHO 1-2x.88meganuclease.

SEQ ID NO: 262 sets forth residues 198-344 of the RHO 1-2x.294meganuclease.

SEQ ID NO: 263 sets forth residues 198-344 of the RHO 1-2x.302meganuclease.

SEQ ID NO: 264 sets forth residues 198-344 of the RHO 1-2x.306meganuclease.

SEQ ID NO: 265 sets forth residues 198-344 of the RHO 1-2x.338meganuclease.

SEQ ID NO: 266 sets forth residues 198-344 of the RHO 1-2x.348meganuclease.

SEQ ID NO: 267 sets forth residues 198-344 of the RHO 1-2x.356meganuclease.

SEQ ID NO: 268 sets forth residues 198-344 of the RHO 1-2x.364meganuclease.

SEQ ID NO: 269 sets forth residues 198-344 of the RHO 1-2x.142meganuclease.

SEQ ID NO: 270 sets forth residues 198-344 of the RHO 1-2x.177meganuclease.

SEQ ID NO: 271 sets forth residues 198-344 of the RHO 1-2x.148meganuclease.

SEQ ID NO: 272 sets forth residues 198-344 of the RHO 1-2x.20meganuclease.

SEQ ID NO: 273 sets forth residues 198-344 of the RHO 1-2x.55meganuclease.

SEQ ID NO: 274 sets forth residues 198-344 of the RHO 1-2x.197meganuclease.

SEQ ID NO: 275 sets forth residues 198-344 of the RHO 1-2x.252meganuclease.

SEQ ID NO: 276 sets forth residues 198-344 of the RHO 1-2x.372meganuclease.

SEQ ID NO: 277 sets forth residues 198-344 of the RHO 1-2x.151meganuclease.

SEQ ID NO: 278 sets forth the nucleic acid sequence of the pDS CMVRHO2_L3_59 plasmid.

SEQ ID NO: 279 sets forth the nucleic acid sequence of the pDS CMVRHO2_L5_14 plasmid.

SEQ ID NO: 280 sets forth the nucleic acid sequence of the pDS GRK1RHO2_L3_59 plasmid.

SEQ ID NO: 281 sets forth the nucleic acid sequence of the pDS GRK1RHO2_L5_14 plasmid.

DETAILED DESCRIPTION OF THE INVENTION 1.1 References and Definitions

The patent and scientific literature referred to herein establishesknowledge that is available to those of skill in the art. The issued USpatents, allowed applications, published foreign applications, andreferences, including GenBank database sequences, which are cited hereinare hereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

The present invention can be embodied in different forms and should notbe construed as limited to the embodiments set forth herein. Rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey the scope of the invention to thoseskilled in the art. For example, features illustrated with respect toone embodiment can be incorporated into other embodiments, and featuresillustrated with respect to a particular embodiment can be deleted fromthat embodiment. In addition, numerous variations and additions to theembodiments suggested herein will be apparent to those skilled in theart in light of the instant disclosure, which do not depart from theinstant invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference herein in their entirety.

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

As used herein, the term “meganuclease” refers to an endonuclease thatbinds double-stranded DNA at a recognition sequence that is greater than12 base pairs. Preferably, the recognition sequence for a meganucleaseof the invention is 22 base pairs. A meganuclease can be an endonucleasethat is derived from I-CreI, and can refer to an engineered variant ofI-CreI that has been modified relative to natural I-CreI with respectto, for example, DNA-binding specificity, DNA cleavage activity,DNA-binding affinity, or dimerization properties. Methods for producingsuch modified variants of I-CreI are known in the art (e.g. WO2007/047859). A meganuclease as used herein binds to double-stranded DNAas a heterodimer. A meganuclease may also be a “single-chainmeganuclease” in which a pair of DNA-binding domains are joined into asingle polypeptide using a peptide linker.

As used herein, the term “single-chain meganuclease” refers to apolypeptide comprising a pair of meganuclease subunits joined by alinker. A single-chain meganuclease has the organization: N-terminalsubunit—Linker—C-terminal subunit. The two meganuclease subunits willgenerally be non-identical in amino acid sequence and will recognizenon-identical DNA sequences. Thus, single-chain meganucleases typicallycleave pseudo-palindromic or non-palindromic recognition sequences. Asingle-chain meganuclease may be referred to as a “single-chainheterodimer” or “single-chain heterodimeric meganuclease” although it isnot, in fact, dimeric. For clarity, unless otherwise specified, the term“meganuclease” can refer to a dimeric or single-chain meganuclease.Methods for producing single—chain meganuclease variants of I-CreI areknown in the art (e.g., WO 2009/059195; Li, et al. (2009) Nucleic AcidsRes. 37:1650-62; Grizot, et al. (2009) Nucleic Acids Res. 37:5405-19).The term “homing endonuclease” is synonymous with the term“meganuclease.”

As used herein, the term “linker” refers to an exogenous peptidesequence used to join two meganuclease subunits into a singlepolypeptide. A linker may have a sequence that is found in naturalproteins, or may be an artificial sequence that is not found in anynatural protein. A linker may be flexible and lacking in secondarystructure or may have a propensity to form a specific three-dimensionalstructure under physiological conditions. A linker can include, withoutlimitation, those encompassed by U.S. Pat. No. 8,445,251. In someembodiments, a linker may have an amino acid sequence comprisingresidues 154-195 of any one of SEQ ID NOs:6-93.

As used herein, with respect to a protein, the term “recombinant” meanshaving an altered amino acid sequence as a result of the application ofgenetic engineering techniques to nucleic acids which encode theprotein, and cells or organisms which express the protein. With respectto a nucleic acid, the term “recombinant” means having an alterednucleic acid sequence as a result of the application of geneticengineering techniques. Genetic engineering techniques include, but arenot limited to, PCR and DNA cloning technologies; transfection,transformation and other gene transfer technologies; homologousrecombination; site-directed mutagenesis; and gene fusion. In accordancewith this definition, a protein having an amino acid sequence identicalto a naturally-occurring protein, but produced by cloning and expressionin a heterologous host, is not considered recombinant.

As used herein, the term “wild-type” refers to the most common naturallyoccurring allele (i.e., polynucleotide sequence) in the allelepopulation of the same type of gene, wherein a polypeptide encoded bythe wild-type allele has its original functions. The term “wild-type”also refers a polypeptide encoded by a wild-type allele. Wild-typealleles (i.e., polynucleotides) and polypeptides are distinguishablefrom mutant or variant alleles and polypeptides, which comprise one ormore mutations and/or substitutions relative to the wild-typesequence(s). Whereas a wild-type allele or polypeptide can confer anormal phenotype in an organism, a mutant or variant allele orpolypeptide can, in some instances, confer an altered phenotype. Themutant RHO P23H allele and the P23H rhodopsin protein aredistinguishable from the wild-type RHO allele and rhodopsin protein.Further, wild-type homing endonucleases are distinguishable fromrecombinant or non-naturally-occurring meganucleases.

As used herein with respect to recombinant proteins, the term“modification” means any insertion, deletion or substitution of an aminoacid residue in the recombinant sequence relative to a referencesequence (e.g., a wild-type or a native sequence).

As used herein, the term “recognition sequence” refers to a DNA sequencethat is bound and cleaved by a meganuclease. In the case of arecombinant meganuclease of the invention, a recognition sequencecomprises a pair of inverted, 9 base pair “half-sites” or “recognitionhalf-sites” which are separated by four base pairs. In the case of asingle-chain meganuclease, the N-terminal subunit of the proteincontacts a first half-site and the C-terminal subunit of the proteincontacts a second half-site. Cleavage by a meganuclease produces fourbase pair 3′ “overhangs”. “Overhangs”, or “sticky ends” are short,single-stranded DNA segments that can be produced by meganucleasecleavage of a double-stranded DNA sequence. In the case of meganucleasesof the present invention, the overhang comprises bases 10-13 of the 22base pair recognition sequence.

As used herein, the term “target site” or “target sequence” refers to aregion of the chromosomal DNA of a cell comprising a recognitionsequence for a meganuclease.

As used herein, the term “DNA-binding affinity” or “binding affinity”means the tendency of a meganuclease to non-covalently associate with areference DNA molecule (e.g., a recognition sequence or an arbitrarysequence). Binding affinity is measured by a dissociation constant,K_(d). As used herein, a meganuclease has “altered” binding affinity ifthe K_(d) of the recombinant meganuclease for a reference recognitionsequence is increased or decreased by a statistically significant(p<0.05) amount relative to a reference meganuclease.

As used herein, the term “homologous recombination” or “HR” refers tothe natural, cellular process in which a double-stranded DNA-break isrepaired using a homologous DNA sequence as the repair template (see,e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologousDNA sequence may be an endogenous chromosomal sequence or an exogenousnucleic acid that was delivered to the cell.

As used herein, the term “non-homologous end-joining” or “NHEJ” refersto the natural, cellular process in which a double-stranded DNA-break isrepaired by the direct joining of two non-homologous DNA segments (see,e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair bynon-homologous end-joining is error-prone and frequently results in theuntemplated addition or deletion of DNA sequences at the site of repair.In some instances, cleavage at a target recognition sequence results inNHEJ at a target recognition site. Nuclease-induced cleavage of a targetsite in the coding sequence of a gene followed by DNA repair by NHEJ canintroduce mutations into the coding sequence, such as frameshiftmutations, that disrupt gene function. Thus, engineered nucleases suchas meganucleases can be used to effectively knock-out a gene in apopulation of cells.

As used herein with respect to both amino acid sequences and nucleicacid sequences, the terms “percent identity,” “sequence identity,”“percentage similarity,” “sequence similarity” and the like refer to ameasure of the degree of similarity of two sequences based upon analignment of the sequences which maximizes similarity between alignedamino acid residues or nucleotides, and which is a function of thenumber of identical or similar residues or nucleotides, the number oftotal residues or nucleotides, and the presence and length of gaps inthe sequence alignment. A variety of algorithms and computer programsare available for determining sequence similarity using standardparameters. As used herein, sequence similarity is measured using theBLASTp program for amino acid sequences and the BLASTn program fornucleic acid sequences, both of which are available through the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov/), and aredescribed in, for example, Altschul et al. (1990), J. Mol. Biol.215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden etal. (1996), Meth. Enzymol 266:131-141; Altschul et al. (1997), NucleicAcids Res. 25:33 89-3402); Zhang et al. (2000), J Comput. Biol.7(1-2):203-14. As used herein, percent similarity of two amino acidsequences is the score based upon the following parameters for theBLASTp algorithm: word size=3; gap opening penalty=−11; gap extensionpenalty=−1; and scoring matrix=BLOSUM62. As used herein, percentsimilarity of two nucleic acid sequences is the score based upon thefollowing parameters for the BLASTn algorithm: word size=11; gap openingpenalty=−5; gap extension penalty=−2; match reward=1; and mismatchpenalty=−3. Similarly, the percent identity can be established basedupon an alignment of the sequences which maximizes identity betweenaligned amino acid residues or nucleotides, and which is a function ofthe number of identical residues or nucleotides divided by the number oftotal residues or nucleotides in the larger of the two sequences.

As used herein with respect to modifications of two proteins or aminoacid sequences, the term “corresponding to” is used to indicate that aspecified modification in the first protein is a substitution of thesame amino acid residue as in the modification in the second protein,and that the amino acid position of the modification in the firstproteins corresponds to or aligns with the amino acid position of themodification in the second protein when the two proteins are subjectedto standard sequence alignments (e.g., using the BLASTp program). Thus,the modification of residue “X” to amino acid “A” in the first proteinwill correspond to the modification of residue “Y” to amino acid “A” inthe second protein if residues X and Y correspond to each other in asequence alignment, and despite the fact that X and Y may be differentnumbers.

As used herein, the term “recognition half-site,” “recognition sequencehalf-site,” or simply “half-site” means a nucleic acid sequence in adouble-stranded DNA molecule which is recognized by a monomer of ahomodimeric or heterodimeric meganuclease, or by one subunit of asingle-chain meganuclease.

As used herein, the term “preferentially” refers to the specificity of arecombinant meganuclease for recognizing and cleaving a particulartarget recognition sequence in the genome relative to a second,reference recognition sequence. By way of example, a recombinantmeganuclease of the invention may preferentially recognize and cleave aP23H recognition sequence (e.g., SEQ ID NO:1) with greater efficiencythan it recognizes and cleaves the corresponding wild-type recognitionsequence (e.g., SEQ ID NO:5), as determined by methods known in the art,including those methods provided in the examples herein. In someembodiments, a recombinant meganuclease of the invention may recognizeand cleave a P23H recognition sequence with greater than about 5%, 10%,15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% moreefficiency than it recognizes and cleaves the corresponding wild-typerecognition sequence. In other embodiments, a recombinant meganucleaseof the invention may recognize and cleave a P23H recognition sequencewith greater than about 1-fold, 2-fold, 5-fold, 10-fold, 50-fold,100-fold, or 1000-fold more efficiency than it recognizes and cleavesthe corresponding wild-type recognition sequence.

As used herein, the term “hypervariable region” refers to a localizedsequence within a meganuclease monomer or subunit that comprises aminoacids with relatively high variability. A hypervariable region cancomprise about 50-60 contiguous residues, about 53-57 contiguousresidues, or preferably about 56 residues. In some embodiments, theresidues of a hypervariable region may correspond to positions 24-79 orpositions 215-270 of any one of SEQ ID NOs:6-93. A hypervariable regioncan comprise one or more residues that contact DNA bases in arecognition sequence and can be modified to alter base preference of themonomer or subunit. A hypervariable region can also comprise one or moreresidues that bind to the DNA backbone when the meganuclease associateswith a double-stranded DNA recognition sequence. Such residues can bemodified to alter the binding affinity of the meganuclease for the DNAbackbone and the target recognition sequence. In different embodimentsof the invention, a hypervariable region may comprise between 1-20residues that exhibit variability and can be modified to influence basepreference and/or DNA-binding affinity. In particular embodiments, ahypervariable region comprises between about 15-18 residues that exhibitvariability and can be modified to influence base preference and/orDNA-binding affinity. In some embodiments, variable residues within ahypervariable region correspond to one or more of positions 24, 26, 28,29, 30, 32, 33, 38, 39, 40, 42, 44, 46, 68, 70, 73, 75, and 77 of anyone of SEQ ID NOs:6-93. In other embodiments, variable residues within ahypervariable region correspond to one or more of positions 215, 217,219, 220, 221, 223, 224, 229, 230, 231, 233, 235, 237, 259, 261, 264,266, and 268 of any one of SEQ ID NOs:6-93.

As used herein, the term “RHO,” “RHO gene,” “rhodopsin gene,” and“wild-type RHO allele” are used interchangeably and refer to the humanrhodopsin gene, preferably the gene identified by NCBI ReferenceSequence NG_009115.1 (SEQ ID NO:98). The terms “mutant RHO allele” and“mutant RHO P23H allele” are used interchangeably and refer to a RHOallele sequence comprising a C68A mutation (SEQ ID NO:99), which resultsin a P23H substitution in the encoded protein. The terms “rhodopsin” and“wild-type rhodopsin” are used interchangeably and refer to the proteinencoded by the wild-type rhodopsin gene, particularly the proteinidentified by NCBI Reference Sequence NP_000530.1 (SEQ ID NO:100). Theterm “P23H rhodopsin” refers to the mutant rhodopsin protein comprisinga P23H substitution, particularly the protein set forth in SEQ IDNO:101.

The terms “recombinant DNA construct,” “recombinant construct,”“expression cassette,” “expression construct,” “chimeric construct,”“construct,” and “recombinant DNA fragment” are used interchangeablyherein and are nucleic acid fragments. A recombinant construct comprisesan artificial combination of nucleic acid fragments, including, withoutlimitation, regulatory and coding sequences that are not found togetherin nature. For example, a recombinant DNA construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source and arranged in a manner different than that foundin nature. Such a construct may be used by itself or may be used inconjunction with a vector.

As used herein, a “vector” or “recombinant DNA vector” may be aconstruct that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. If a vector is used then the choice ofvector is dependent upon the method that will be used to transform hostcells as is well known to those skilled in the art. Vectors can include,without limitation, plasmid vectors and recombinant AAV vectors, or anyother vector known in that art suitable for delivering a gene encoding ameganuclease of the invention to a target cell. The skilled artisan iswell aware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleotides or nucleic acid sequences ofthe invention.

As used herein, a “vector” can also refer to a viral vector. Viralvectors can include, without limitation, retroviral vectors, lentiviralvectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

As used herein, a “target cell” refers to a cell that comprises at leastone RHO allele comprising a P23H recognition sequence (e.g., one of SEQID NOs:1-4). Such target cells can express mutant RHO P23H protein.Target cells can include, without limitation, cells of the eye,preferably cells in the posterior segment of the eye, and even morepreferably cells of the retina, including rod photoreceptor cellscomprising a P23H recognition sequence in at least one RHO gene allele.

As used herein, a “control” or “control cell” refers to a cell thatprovides a reference point for measuring changes in genotype orphenotype of a genetically-modified cell. A control cell may comprise,for example: (a) a wild-type cell, i.e., of the same genotype as thestarting material for the genetic alteration which resulted in thegenetically-modified cell; (b) a cell of the same genotype as thegenetically-modified cell but which has been transformed with a nullconstruct (i.e., with a construct which has no known effect on the traitof interest); or, (c) a cell genetically identical to thegenetically-modified cell but which is not exposed to conditions orstimuli or further genetic modifications that would induce expression ofaltered genotype or phenotype.

As used herein with respect to modifications of two proteins or aminoacid sequences, the term “corresponding to” is used to indicate that aspecified modification in the first protein is a substitution of thesame amino acid residue as in the modification in the second protein,and that the amino acid position of the modification in the firstproteins corresponds to or aligns with the amino acid position of themodification in the second protein when the two proteins are subjectedto standard sequence alignments (e.g., using the BLASTp program). Thus,the modification of residue “X” to amino acid “A” in the first proteinwill correspond to the modification of residue “Y” to amino acid “A” inthe second protein if residues X and Y correspond to each other in asequence alignment, and despite the fact that X and Y may be differentnumbers.

As used herein, the terms “treatment” or “treating a subject” refers tothe administration of recombinant meganuclease of the invention, or anucleic acid encoding recombinant meganuclease of the invention, to asubject having RP for the purpose of providing partial or completerelief of one or more symptoms of RP. In some aspects, recombinantmeganuclease of the invention, or a nucleic acid encoding the same, isadministered during treatment in the form of a pharmaceuticalcomposition of the invention. Preferably the subject is heterozygous forthe P23H mutant allele and a functional, normal or wild-type allele.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable can be equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variablecan be equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 can takethe values 0, 1 or 2 if the variable is inherently discrete, and cantake the values 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and≤2 if the variable is inherently continuous.

2.1 Principle of Targeting and Inactivating the Mutant RHO P23H Allele

The present invention is based, in part, on the hypothesis thatautosomal dominant RP can be corrected or prevented by targeting,cleaving, and inactivating a mutant RHO P23H allele, which encodes thepathogenic P23H rhodopsin protein. Surprisingly, recombinantmeganucleases can be engineered to recognize and cleave a P23Hrecognition sequence (e.g., one of SEQ ID NOs:1-4) present in the mutantRHO P23H allele. Such recombinant meganucleases can preferentiallytarget and cleave the mutant RHO P23H allele relative to thecorresponding wild-type allele (SEQ ID NO:96). NHEJ at the cleavage siteresults in mutagenesis and disruption of the mutant RHO P23H allele,while the functional wild-type RHO allele remains intact to expresswild-type rhodopsin in rod photoreceptor cells of the retina.Preferential inactivation of the mutant RHO P23H allele, and disruptionof P23H rhodopsin expression, is expected to prevent, delay, or reversethe progression of RP in patients.

2.2 Meganucleases for Recognizing and Cleaving the P23H RecognitionSequence

Meganucleases can make a site-specific DNA break in the genome of aliving cell, and such a DNA break can result in permanent modificationof the genome via mutagenic NHEJ repair or via homologous recombinationwith a transgenic DNA sequence. NHEJ can produce mutagenesis at thecleavage site, resulting in inactivation of the allele. NHEJ-associatedmutagenesis may inactivate an allele via generation of early stopcodons, frameshift mutations producing aberrant non-functional proteins,or could trigger mechanisms such as nonsense-mediated mRNA decay. Theuse of meganucleases to induce mutagenesis via NHEJ can be used totarget a specific mutation or a sequence present in a wild-type allele.

In preferred embodiments, the nucleases used to practice the inventionare single-chain meganucleases. A single-chain meganuclease comprises anN-terminal subunit and a C-terminal subunit joined by a linker peptide.Each of the two domains recognizes half of the recognition sequence(i.e., a recognition half-site) and the site of DNA cleavage is at themiddle of the recognition sequence near the interface of the twosubunits. DNA strand breaks are offset by four base pairs such that DNAcleavage by a meganuclease generates a pair of four base pair, 3′single-strand overhangs.

Recombinant meganucleases of the invention have been engineered torecognize and cleave a P23H recognition sequence (e.g., one of SEQ IDNOs:1-4). Such meganucleases preferentially cleave the P23H recognitionsequence on the mutant RHO P23H allele relative to the correspondingwild-type RHO recognition sequence (SEQ ID NO:96). Exemplary recombinantmeganucleases of the invention are provided in SEQ ID NOs:6-93, whichare collectively referred to herein as “RHO 1-2 meganucleases” or “RHO1/2 meganucleases.

Recombinant meganucleases of the invention comprise a first subunit,comprising a first hypervariable (HVR1) region, and a second subunit,comprising a second hypervariable (HVR2) region. Further, the firstsubunit binds to a first recognition half-site in a P23H recognitionsequence (e.g., the RHO1 half-site), and the second subunit binds to asecond recognition half-site in the P23H recognition sequence (e.g., theRHO2 half-site). In embodiments where the recombinant meganuclease is asingle-chain meganuclease, the first and second subunits can be orientedsuch that the first subunit is positioned as the N-terminal subunit, andthe second subunit is positioned as the C-terminal subunit (e.g., SEQ IDNOs:70-93). In alternative embodiments, the first and second subunitscan be oriented such that the first subunit is positioned as theC-terminal subunit, and the second subunit is positioned as theN-terminal subunit (e.g., SEQ ID NOs:6-69). Exemplary recombinantmeganucleases of the invention are provided in Table 1.

TABLE 1 Exemplary recombinant meganucleases engineered to recognize andcleave the P23H recognition sequence (SEQ ID NO: 1) RHO1 RHO1 RHO2 RHO2AA Subunit Subunit *RHO1 Subunit Subunit *RHO2 Meganuelease SEQ IDResidues SEQ ID Subunit % Residues SEQ ID Subunit % RHO2-L3-59 6 198-344102 100  7-153 187 100 RHO2-L5-14 7 198-344 103 100  7-153 188 96.6RHO-1/2-L2-49 8 198-344 104 100  7-153 189 96.6 RHO 1-2x.179 9 198-344105 100  7-153 190 93.2 RHO 1-2x.4 10 198-344 106 92.52  7-153 191 91.16RHO 1-2x.207 11 198-344 107 93.2  7-153 192 91.84 RHO 1-2x.277 12198-344 108 93.2  7-153 193 91.84 RHO 1-2x.292 13 198-344 109 94.56 7-153 194 90.48 RHO 1-2x.324 14 198-344 110 92.52  7-153 195 91.16 RHO1-2x.371 15 198-344 111 93.2  7-153 196 91.16 RHO 1-2x.164 16 198-344112 91.16  7-153 197 90.48 RHO 1-2x.181 17 198-344 113 95.24  7-153 19890.48 RHO 1-2x.184 18 198-344 114 90.48  7-153 199 88.44 RHO-1/2-L1-2119 198-344 115 100  7-153 200 93.2 RHO-1/2-L1-43 20 198-344 116 100 7-153 201 92.52 RHO-1/2-L1-45 21 198-344 117 100  7-153 202 92.52RHO-1/2-L1-60 22 198-344 118 100  7-153 203 92.52 RHO-1/2-L1-61 23198-344 119 100  7-153 204 92.52 RHO-1/2-L1-58 74 198-344 120 100  7-153205 92.52 RHO-1/2-L1-7 25 198-344 121 100  7-153 206 92.52 RHO-1/2-L1-1326 198-344 122 100  7-153 207 92.52 RHO-1/2-L1-18 27 198-344 123 100 7-153 208 92.52 RHO-1/2-L1-70 28 198-344 124 100  7-153 209 92.52RHO-1/2-L1-86 29 198-344 125 100  7-153 210 92.52 RHO-1/2-L2-13 30198-344 126 100  7-153 211 95.24 RHO-1/2-L2-24 31 198-344 127 100  7-153212 95.24 RHO-1/2-L2-37 32 198-344 128 100  7-153 213 93.88RHO-1/2-L2-58 33 198-344 129 100  7-153 214 93.2 RHO-1/2-L2-31 34198-344 130 100  7-153 215 93.2 RHO-1/2-L2-29 35 198-344 131 100  7-153216 93.2 RHO-1/2-L2-61 36 198-344 132 100  7-153 217 93.88 RHO2-L3-2 37198-344 133 100  7-153 218 94.56 RHO2-L3-3 38 198-344 134 100  7-153 21995.24 RHO2-L3-5 39 198-344 135 100  7-153 220 96.6 RHO2-L3-10 40 198-344136 100  7-153 221 94.56 RHO2-L3-11 41 198-344 137 100  7-153 222 95.24RHO2-L3-12 42 198-344 138 100  7-153 223 95.24 RHO2-L3-13 43 198-344 139100  7-153 224 95.24 RHO2-L3-28 44 198-344 140 100  7-153 225 95.24RHO2-L3-29 45 198-344 141 100  7-153 226 94.56 RHO2-L3-57 46 198-344 142100  7-153 227 95.24 RHO2-L3-80 47 198-344 143 100  7-153 228 95.92RHO2-L3-85 48 198-344 144 100  7-153 229 95.24 RHO2-L3-86 49 198-344 145100  7-153 230 94.56 RHO2-L3-92 50 198-344 146 100  7-153 231 95.24RHO2-L3-4 51 198-344 147 100  7-153 232 94.56 RHO2-L3-20 52 198-344 148100  7-153 233 94.56 RHO2-L3-72 53 198-344 149 100  7-153 234 95.92RHO1-L1-4 54 198-344 150 96.6  7-153 235 96.6 RHO1-L1-8 55 198-344 15195.24  7-153 236 96.6 RHO1-L1-13 56 198-344 152 95.24  7-153 237 96.6RHO1-L1-19 57 198-344 153 95.24  7-153 238 96.6 RHO1-L1-58 58 198-344154 95.92  7-153 239 96.6 RHO1-L1-69 59 198-344 155 95.92  7-153 24096.6 RHO1-L1-80 60 198-344 156 95.92  7-153 241 96.6 RHO1-L1-82 61198-344 157 95.92  7-153 245 96.6 RHO1-L1-73 62 198-344 158 96.6  7-153246 96.6 RHO1-L1-85 63 198-344 159 96.6  7-153 247 96.6 RHO1-L1-86 64198-344 160 96.6  7-153 248 96.6 RHO-1/2-L4-10 65 198-344 161 95.24 7-153 249 95.24 RHO-1/2-L4-29 66 198-344 162 95.24  7-153 250 95.24RHO-1/2-L4-65 67 198-344 163 95.92  7-153 251 94.56 RHO-1/2-L4-66 68198-344 164 97.28  7-153 252 95.24 RHO-1/2-L4-85 69 198-344 165 96.6 7-153 253 94.56 RHO 1-2x.216 70  7-153 166 93.2 198-344 254 91.16 RHO1-2x.241 71  7-153 167 93.88 198-344 255 91.16 RHO 1-2x.94 72  7-153 16891.84 198-344 256 91.16 RHO 1-2x.95 73  7-153 169 91.16 198-344 25791.16 RHO 1-2x.1 74  7-153 170 93.2 198-344 258 90.48 RHO 1-2x.60 75 7-153 171 93.2 198-344 259 90.48 RHO 1-2x.74 76  7-153 172 93.88198-344 260 90.48 RHO 1-2x.88 77  7-153 173 91.16 198-344 261 90.48 RHO1-2x.294 78  7-153 174 93.88 198-344 262 90.48 RHO 1-2x.302 79  7-153175 93.2 198-344 263 90.48 RHO 1-2x.306 80  7-153 176 92.52 198-344 26490.48 RHO 1-2x.338 81  7-153 177 93.2 198-344 265 90.48 RHO 1-2x.348 82 7-153 178 92.52 198-344 266 90.48 RHO 1-2x.356 83  7-153 179 91.16198-344 267 90.48 RHO 1-2x.364 84  7-153 180 93.2 198-344 268 90.48 RHO1-2x.142 85  7-153 181 93.2 198-344 269 90.48 RHO 1-2x.177 86  7-153 18294.56 198-344 270 90.48 RHO 1-2x.148 87  7-153 183 91.84 198-344 27193.2 RHO 1-2x.20 88  7-153 184 93.2 198-344 272 89.12 RHO 1-2x.55 89 7-153 185 92.52 198-344 273 92.52 RHO 1-2x.197 90  7-153 186 95.24198-344 274 91.84 RHO 1-2x.252 91  7-153 187 93.2 198-344 275 88.44 RHO1-2x.372 92  7-153 188 94.56 198-344 276 91.84 RHO 1-2x.151 93  7-153189 94.56 198-344 277 90.48 *“RHO1 Subunit %” and “RHO2 Subunit %”represent the amino acid sequence identity between the RHO1-binding andRHO2-binding subunit regions of each meganuclease and the RHO1-bindingand RHO2-binding subunit regions, respectively, of the RHO2-L3-59meganuclease.

2.3 Methods for Delivering and Expressing Recombinant Meganucleases

Treating RP using the invention requires that a recombinant meganucleasecan be expressed in cells in the appropriate tissues. The targettissue(s) for delivery of recombinant meganucleases of the invention arecells of the eye, preferably cells in the posterior segment of the eye,and even more preferably cells of the retina, including rodphotoreceptor cells. Recombinant meganucleases can be delivered aspurified protein or as RNA or DNA encoding the meganucleases. In oneembodiment, recombinant meganuclease proteins, or mRNA or vectorencoding recombinant meganucleases, are supplied to target cells (e.g.,cells in the retina) via injection directly to the target tissue. Forexample, delivery of RNA, DNA, or recombinant AAV vectors to the eye viasubretinal or intravitreal injection is described in the art (see forexample, Martin et al. (2002) Methods. 28:267-275; Hauswirth et al.(2008) Human Gene Therapy 19(10):979-990; Johnson et al. (2008)Molecular Vision. 14:2211-2226). Alternatively, meganuclease protein,mRNA, or DNA can be delivered systemically via the circulatory system.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are formulated for systemicadministration, or administration to target tissues, in a pharmaceuticalcarrier in accordance with known techniques. See, e.g., Remington, TheScience And Practice of Pharmacy (20 ed. 2005 the manufacture of apharmaceutical formulation according to the invention, proteins/RNA/mRNAare typically admixed with a pharmaceutically acceptable carrier. Thecarrier must, of course, be acceptable in the sense of being compatiblewith any other ingredients in the formulation and must not bedeleterious to the patient. The carrier can be a solid or a liquid, orboth, and can be formulated with the compound as a unit-doseformulation.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are coupled to a cell penetratingpeptide or targeting ligand to facilitate cellular uptake. Examples ofcell penetrating peptides known in the art include poly-arginine(Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptidefrom the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25: 679-736),MPG (Simeoni, et al. (2003) Nucleic Acids Res. 31:2717-2724), Pep-1(Deshayes et al. (2004) Biochemistry 43: 7698-7706, and HSV-1 VP-22(Deshayes et al. (2005) Cell Mol Life Sci. 62:1839-49. In an alternativeembodiment, recombinant meganucleases, or DNA/mRNA encoding recombinantmeganucleases, are coupled covalently or non-covalently to an antibodythat recognizes a specific cell-surface receptor expressed on targetcells such that the meganuclease protein/DNA/mRNA binds to and isinternalized by the target cells. Alternatively, recombinantmeganuclease protein/DNA/mRNA can be coupled covalently ornon-covalently to the natural ligand (or a portion of the naturalligand) for such a cell-surface receptor. (McCall, et al. (2014) TissueBarriers. 2(4):e944449; Dinda, et al. (2013) Curr Pharm Biotechnol.14:1264-74; Kang, et al. (2014) Curr Pharm Biotechnol. 15(3):220-30;Qian et al. (2014) Expert Opin Drug Metab Toxicol. 10(11):1491-508).Examples of targeting ligands to direct delivery to cells in the eyeinclude RGD (Pullinger et al. (2013) PNAS. 110(15): 6115-6120),transferrin (Lajunen et al. (2014) Eur J Pharm Sci. 62: 23-32), andhyaluronic acid (Martens et al. (2015) J Control Release. 202: 83-92).

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are encapsulated withinbiodegradable hydrogels for injection or implantation within the desiredregion of the eye (e.g., intravitreal or subconjunctival injection).Hydrogels can provide sustained and tunable release of the therapeuticpayload to the desired region of the eye without the need for frequentinjections, and stimuli-responsive materials (e.g., temperature- andpH-responsive hydrogels) can be designed to release the payload inresponse to environmental or externally applied cues (Kang Derwent etal. (2008) Trans Am Ophthalmol Soc. 106:206-214).

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are coupled covalently or,preferably, non-covalently to a nanoparticle or encapsulated within sucha nanoparticle using methods known in the art (Sharma, et al. (2014)Biomed Res Int. 2014). A nanoparticle is a nanoscale delivery systemwhose length scale is <1 μm, preferably <100 nm. Such nanoparticles maybe designed using a core composed of metal, lipid, polymer, orbiological macromolecule, and multiple copies of the recombinantmeganuclease proteins, mRNA, or DNA can be attached to or encapsulatedwith the nanoparticle core. This increases the copy number of theprotein/mRNA/DNA that is delivered to each cell and, so, increases theintracellular expression of each recombinant meganuclease to maximizethe likelihood that the target recognition sequences will be cut. Thesurface of such nanoparticles may be further modified with polymers orlipids (e.g., chitosan, cationic polymers, or cationic lipids) to form acore-shell nanoparticle whose surface confers additional functionalitiesto enhance cellular delivery and uptake of the payload (Jian et al.(2012), Biomaterials. 33(30): 7621-30). Nanoparticles may additionallybe advantageously coupled to targeting molecules to direct thenanoparticle to the appropriate cell type and/or increase the likelihoodof cellular uptake. Examples of such targeting molecules includeantibodies specific for cell-surface receptors and the natural ligands(or portions of the natural ligands) for cell surface receptors.

In some embodiments, the meganuclease proteins or DNA/mRNA encoding themeganucleases are encapsulated within liposomes or complexed usingcationic lipids (see, e.g., Lipofectamine™, Life Technologies Corp.,Carlsbad, Calif.; Zuris et al. (2015), Nat Biotechnol. 33: 73-80; Mishraet al. (2011), J Drug Deliv. 2011:863734). The liposome and lipoplexformulations can protect the payload from degradation, enhanceaccumulation and retention at the target site, and facilitate cellularuptake and delivery efficiency through fusion with and/or disruption ofthe cellular membranes of the target cells.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are encapsulated within polymericscaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI,PLL) (Tamboli et al. (2011), Ther Deliv. 2(4): 523-536). Polymericcarriers can be designed to provide tunable drug release rates throughcontrol of polymer erosion and drug diffusion, and high drugencapsulation efficiencies can offer protection of the therapeuticpayload until intracellular delivery to the desired target cellpopulation.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are combined with amphiphilicmolecules that self-assemble into micelles (Tong et al. (2007), J GeneMed. 9(11): 956-66). Polymeric micelles may include a micellar shellformed with a hydrophilic polymer (e.g., polyethyleneglycol) that canprevent aggregation, mask charge interactions, and reduce nonspecificinteractions within the vitreous fluid.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are formulated into an emulsion or ananoemulsion (i.e., having an average particle diameter of <1 nm) foradministration and/or delivery to the target cell. The term “emulsion”refers to, without limitation, any oil-in-water, water-in-oil,water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets,including lipid structures that can form as a result of hydrophobicforces that drive apolar residues (e.g., long hydrocarbon chains) awayfrom water and polar head groups toward water, when a water immisciblephase is mixed with an aqueous phase. These other lipid structuresinclude, but are not limited to, unilamellar, paucilamellar, andmultilamellar lipid vesicles, micelles, and lamellar phases. Emulsionsare composed of an aqueous phase and a lipophilic phase (typicallycontaining an oil and an organic solvent). Emulsions also frequentlycontain one or more surfactants. Nanoemulsion formulations are wellknown, e.g., as described in US Patent Application Nos. 2002/0045667 and2004/0043041, and U.S. Pat. Nos. 6,015,832, 6,506,803, 6,635,676, and6,559,189, each of which is incorporated herein by reference in itsentirety.

In some embodiments, recombinant meganuclease proteins, or DNA/mRNAencoding recombinant meganucleases, are covalently attached to, ornon-covalently associated with, multifunctional polymer conjugates, DNAdendrimers, and polymeric dendrimers (Mastorakos et al. (2015),Nanoscale. 7(9): 3845-56; Cheng et al. (2008), J Pharm Sci. 97(1):123-43). The dendrimer generation can control the payload capacity andsize, and can provide a high drug payload capacity. Moreover, display ofmultiple surface groups can be leveraged to improve stability, reducenonspecific interactions, and enhance cell-specific targeting and drugrelease.

In some embodiments, genes encoding a recombinant meganuclease aredelivered using a viral vector. Such vectors are known in the art andinclude lentiviral vectors, adenoviral vectors, and adeno-associatedvirus (AAV) vectors (reviewed in Vannucci, et al. (2013), New Microbiol.36:1-22). In some embodiments, the viral vectors are injected directlyinto target tissues (Bosch, et al. (2000), Mol Ther. 1:63-70; Greig etal. (2014), PLoS One. November 13; 9(11):e112268). In alternativeembodiments, the viral vectors are delivered systemically via thecirculatory system. It is known in the art that different AAV vectorstend to localize to different tissues. In retinal target tissues,effective transduction of retinal photoreceptor cells has been shown,for example, with AAV serotypes 1, 2, 5, 8, and 9 (Petrs-Silva et al.(2014), Clinical Ophthalmology. 8:127-136). AAV vectors can also beself-complementary such that they do not require second-strand DNAsynthesis in the host cell (McCarty et al. (2001), Gene Ther.8:1248-54).

In one embodiment, a viral vector used for meganuclease gene delivery isa self-limiting viral vector. A self-limiting viral vector can havelimited persistence time in a cell or organism due to the presence of arecognition sequence for a recombinant meganuclease within the vector.Thus, a self-limiting viral vector can be engineered to provide codingfor a promoter, a recombinant meganuclease described herein, and ameganuclease recognition site within the ITRs. The self-limiting viralvector delivers the meganuclease gene to a cell, tissue, or organism,such that the meganuclease is expressed and able to cut the genome ofthe cell at an endogenous recognition sequence within the genome. Thedelivered meganuclease will also find its target site within theself-limiting viral vector itself, and cut the vector at this targetsite. Once cut, the 5′ and 3′ ends of the viral genome will be exposedand degraded by exonucleases, thus killing the virus and ceasingproduction of the recombinant meganuclease.

If the recombinant meganuclease genes are delivered in DNA form (e.g.plasmid) and/or via a viral vector (e.g. AAV) they must be operablylinked to a promoter. In some embodiments, this can be a viral promotersuch as endogenous promoters from the viral vector (e.g. the LTR of alentiviral vector) or the well-known cytomegalovirus- or SV40virus-early promoters. In a preferred embodiment, meganuclease genes areoperably linked to a promoter that drives gene expression preferentiallyin the target cells. Examples of retina and/or rod photoreceptorcell-specific promoters include, without limitation, the human rhodopsinkinase promoter, the proximal mouse opsin promoter (mOP), the humanG-protein-coupled receptor protein kinase 1 promoter (hGRK1), and thehuman interphotoreceptor retinoid-binding protein (IRBP) promoter (Khaniet al. (2007), Invest. Ophthamol. Vis. Sci. 48(9):3954-3961); Beltran etal. (2010), Gene Therapy. 17(9):1162-1174); Yokoyama et al. (1992), Exp.Eye Res. 55(2):225-233), as well as rod photoreceptor cell-specificpromoters disclosed in U.S. Patent Publication No. US 2014/0287510.

In some embodiments, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a recombinantmeganuclease of the invention, or a pharmaceutically acceptable carrierand an isolated polynucleotide comprising a nucleic acid encoding arecombinant meganuclease of the invention. Such pharmaceuticalcompositions can be prepared in accordance with known techniques. See,e.g., Remington, The Science And Practice of Pharmacy (21^(st) ed.2005). In the manufacture of a pharmaceutical formulation according tothe invention, endonuclease polypeptides (or DNA/RNA encoding the same)are typically admixed with a pharmaceutically acceptable carrier and theresulting composition is administered to a subject. The carrier must, ofcourse, be acceptable in the sense of being compatible with any otheringredients in the formulation and must not be deleterious to thesubject. In some embodiments, pharmaceutical compositions of theinvention can further comprise one or more additional agents orbiological molecules useful in the treatment of a disease in thesubject. Likewise, the additional agent(s) and/or biological molecule(s)can be co-administered as a separate composition.

It is envisioned that a single treatment will permanently inactivate themutant P23H RHO allele in a percentage of patient target cells. If thefrequency of P23H allele inactivation is low, however, or if a largepercentage of target cells need to be corrected, it may be necessary toperform in multiple treatments on each patient.

2.4 Recombinant Meganuclease Variants

Embodiments of the invention encompass the recombinant meganucleasesdescribed herein, and variants thereof. Further embodiments of theinvention encompass isolated polynucleotides comprising a nucleic acidsequence encoding the recombinant meganucleases described herein, andvariants of such polynucleotides.

In particular, the invention provides variants of the meganucleases ofSEQ ID Nos:6-93 in which the hypervariable regions have been modifiedsuch that the meganucleases preferentially recognize and cleave one ofSEQ ID NOs: 2-4 relative to the corresponding wild-type sequences in thewild-type RHO gene (SEQ ID NO: 98). Such additional variantsmeganucleases can be produced by routine experimentation according tothe methods described in WO2007/047859, U.S. Pat. Nos. 8,021,867,8,119,361, 8,119,381, 8,124,369, 8,129,134, 8,133,697, 8,143,015,8,143,016, 8,148,098, 8,163,514, 8,304,222, 8,377,674 and 8,445,251, anddiscussed below.

As used herein, “variants” is intended to mean substantially similarsequences. A “variant” polypeptide is intended to mean a polypeptidederived from the “native” polypeptide by deletion or addition of one ormore amino acids at one or more internal sites in the native proteinand/or substitution of one or more amino acids at one or more sites inthe native polypeptide. As used herein, a “native” polynucleotide orpolypeptide comprises a parental sequence from which variants arederived. Variant polypeptides encompassed by the embodiments arebiologically active. That is, they continue to possess the desiredbiological activity of the native protein; i.e., the ability topreferentially recognize and cleave a P23H recognition sequence (e.g.,one of SEQ II) NOs:1-4), as described herein. Such variants may result,for example, from human manipulation. Biologically active variants of anative polypeptide of the embodiments (e.g., SEQ ID NOs:6-93), orbiologically active variants of the RHO1- and RHO2-binding subunitsdescribed herein (e.g., SEQ ID NOs:99-274), will have at least about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about99%, sequence identity to the amino acid sequence of the nativepolypeptide or native subunit, as determined by sequence alignmentprograms and parameters described elsewhere herein. A biologicallyactive variant of a polypeptide or subunit of the embodiments may differfrom that polypeptide or subunit by as few as about 1-40 amino acidresidues, as few as about 1-20, as few as about 1-10, as few as about 5,as few as 4, 3, 2, or even 1 amino acid residue.

The polypeptides of the embodiments may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants can be prepared bymutations in the DNA. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, for example, Kunkel (1985),Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987), Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983), Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978),Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be optimal.

A substantial number of amino acid modifications to the DNA recognitiondomain of the wild-type I-CreI meganuclease have previously beenidentified (e.g., U.S. Pat. No. 8,021,867) which, singly or incombination, result in recombinant meganucleases with specificitiesaltered at individual bases within the DNA recognition sequencehalf-site, such that the resulting rationally-designed meganucleaseshave half-site specificities different from the wild-type enzyme. Table2 provides potential substitutions that can be made in a recombinantmeganuclease monomer or subunit to enhance specificity based on the basepresent at each half-site position (-1 through -9) of a recognitionhalf-site.

TABLE 2 Favored Sense-Strand Base Posn. A C G T A/T A/C A/G C/T G/TA/G/T A/C/G/T −1 Y75 R70* K70 Q70* T46* G70 L75* H75* E70* C70 A70 C75*R75* E75* L70 S70 Y139* H46* E46* Y75* G46* C46* K46* D46* Q75* A46*R46* H75* H139 Q46* H46* −2 Q70 E70 H70 Q44* C44* T44* D70 D44* A44*K44* E44* V44* R44* I44* L44* N44* −3 Q68 E68 R68 M68 H68 Y68 K68 C24*F68 C68 I24* K24* L68 R24* F68 −4 A26* E77 R77 S77 S26* Q77 K26* E26*Q26* −5 E42 R42 K28* C28* M66 Q42 K66 −6 Q40 E40 R40 C40 A40 S40 C28*R28* I40 A79 S28* V40 A28* C79 H28* I79 V79 Q28* −7 N30* E38 K38 I38 C38H38 Q38 K30* R38 L38 N38 R30* E30* Q30* −8 F33 E33 F33 L33 R32* R33 Y33D33 H33 V33 I33 F33 C33 −9 E32 R32 L32 D32 S32 K32 V32 I32 N32 A32 H32C32 Q32 T32

For polynucleotides, a “variant” comprises a deletion and/or addition ofone or more nucleotides at one or more sites within the nativepolynucleotide. One of skill in the art will recognize that variants ofthe nucleic acids of the embodiments will be constructed such that theopen reading frame is maintained. For polynucleotides, conservativevariants include those sequences that, because of the degeneracy of thegenetic code, encode the amino acid sequence of one of the polypeptidesof the embodiments. Variant polynucleotides include syntheticallyderived polynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a recombinantmeganuclease of the embodiments. Generally, variants of a particularpolynucleotide of the embodiments will have at least about 40%, about45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99% or moresequence identity to that particular polynucleotide as determined bysequence alignment programs and parameters described elsewhere herein.Variants of a particular polynucleotide of the embodiments (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the polypeptide. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by screening the polypeptide for its ability topreferentially recognize and cleave a P23H recognition sequence (e.g.,one of SEQ ID NOs:1-4).

EXAMPLES

This invention is further illustrated by the following examples, whichshould not be construed as limiting. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific substances andprocedures described herein. Such equivalents are intended to beencompassed in the scope of the claims that follow the examples below.

Example 1 Evaluation of Meganucleases that Recognize and Cleave a P23HRecognition Sequence

1. Meganucleases that Recognize and Cleave a P23H Recognition Sequence

Recombinant meganucleases (SEQ ID NOs:6-93), collectively referred toherein as “RHO 1-2 meganucleases,” were engineered to recognize andcleave one of the P23H recognition sequences (i.e., SEQ ID NO:1), whichis present in the mutant RHO P23H allele (see, FIG. 1A). Each RHO 1-2recombinant meganuclease comprises an N-terminal nuclease-localizationsignal derived from SV40, a first meganuclease subunit, a linkersequence, and a second meganuclease subunit. A first subunit in each RHO1-2 meganuclease binds to the RHO1 recognition half-site of SEQ ID NO:1,while a second subunit binds to the RHO2 recognition half-site (see,FIG. 1B).

As illustrated in FIGS. 2 and 3, RHO1-binding subunits and RHO2-bindingsubunits each comprise a 56 base pair hypervariable region, referred toas HVR1 and HVR2, respectively. RHO1-binding subunits are identicaloutside of the HVR1 region except at position 80 or position 271(comprising a Q or E residue), and are highly conserved within the HVR1region. Similarly, RHO2-binding subunits are also identical outside ofthe HVR2 region except at position 80 or position 271 (comprising a Q orE residue), and are highly conserved within the HVR2 region.

The RHO1-binding regions of SEQ ID NOs:6-93 are illustrated in FIGS.2A-2F and are provided as SEQ ID NOs:102-189, respectively. Nearly allof SEQ ID NOs:102-189 share at least 90% sequence identity to SEQ IDNO:102, which is the RHO1-binding region of the meganucleaseRI-102-L3-59 (SEQ ID NO:6). RHO2-binding regions of SEQ ID NOs:6-93 areillustrated in FIGS. 3A-3F and are provided as SEQ ID NOs:190-277,respectively. Nearly all of SEQ ID NOs:190-277 share at least 90%sequence identity to SEQ ID NO:190, which is the RHO2-binding region ofthe meganuclease RHO2-L3-59 (SEQ 1D NO:6).

2. Cleavage of a P23H Recognition Sequence in a CHO Cell Reporter Assay

To determine whether RHO 1-2 meganucleases could recognize and cleavethe P23H recognition sequence of SEQ ID NO:1, each RHO 1-2 meganucleasewas evaluated using the CHO cell reporter assay previously described(see WO2012/167192, FIG. 4). To perform the assay, a pair of CHO cellreporter lines were produced which carried a non-functional GreenFluorescent Protein (GFP) gene expression cassette integrated into thegenome of the cell. The GFP gene in each cell line was interrupted by apair of recognition sequences such that intracellular cleavage of eitherrecognition sequence by a meganuclease would stimulate a homologousrecombination event resulting in a functional GFP gene. In both celllines, one of the recognition sequences was derived from the RHO gene(either SEQ ID NO:1 or SEQ ID NO:5) and the second recognition sequencewas specifically recognized by a control meganuclease called“CHO-23/24”. CHO reporter cells comprising the P23H recognition sequenceof SEQ ID NO:1 and the CHO-23/24 recognition sequence are referred toherein as “RHO 1-2 cells.” RHO 1-2 cells were transfected with plasmidDNA encoding one of the RHO 1-2 meganucleases (SEQ ID NOs:6-93) orencoding the CHO-23/34 meganuclease. Approximately 4×10⁵ CHO cells weretransfected with 50 ng of plasmid DNA in a 96-well plate usingLipofectamine 2000 (ThermoFisher) according to the manufacturer'sinstructions. At 48 hours post-transfection, cells were evaluated byflow cytometry to determine the percentage of GFP-positive cellscompared to an untransfected negative control (RHO 1-2 bs). All RHO 1-2meganucleases were found to produce GFP-positive cells in cell linescomprising the P23H recognition sequence at frequencies significantlyexceeding the negative control and comparable to or exceeding theCHO-23/24 positive control, indicating that each RHO 1-2 meganucleasewas able to efficiently recognize and cleave the intended P23Hrecognition sequence in a cell (see, FIG. 5).

The efficacy of RHO 1-2 recombinant meganucleases was also determined ina time-dependent manner 1, 4, 6, and 8 days after introduction of themeganucleases into RHO 1-2 cells. In this study, RHO 1-2 cells (1.0×10⁶)were electroporated with 1×10⁶ copies of meganuclease mRNA per cellusing a BioRad Gene Pulser Xcell according to the manufacturer'sinstructions. At 48 hours post-transfection, cells were evaluated byflow cytometry to determine the percentage of GFP-positive cells. ACHO-23/24 meganuclease was also included at each time point as apositive control. As shown in FIG. 6, the efficacy of RHO 1-2meganucleases persisted over the 8 day period evaluated.

3. Conclusions

These studies demonstrated that RHO 1-2 meganucleases (SEQ ID NOs:6-93)encompassed by the invention can target and cleave the P23H recognitionsequence of SEQ ID NO:1 in cells.

Example 2 Specificity of Meganucleases for a P23H RecognitionSequence 1. CHO Reporter Cells Comprising a Corresponding Wild-Type RHOSequence

To determine the specificity of RHO 1-2 meganucleases (SEQ ID NOs:6-93)for the P23H recognition sequence of SEQ ID NO:1, a CHO reporter cellline was generated as previously described that comprises thecorresponding wild-type RHO recognition sequence (SEQ ID NO:5), referredto herein as the “RHO 3-4 recognition sequence,” and the CHO-23/24recognition sequence. The resulting cells are referred to herein as “RHO3-4 cells.”

2. Specificity of RHO 1-2 Meganucleases

RHO 1-2 meganucleases were introduced into RHO 1-2 cells or RHO 3-4cells to determine if the recombinant meganucleases could discriminatebetween the P23H recognition sequence of SEQ ID NO:1 and thecorresponding wild-type recognition sequence (SEQ ID NO:5). RHO 1-2meganuclease mRNA was introduced into RHO 1-2 cells or RHO 3-4 cells viaelectroporation of mRNA as previously described, and the percentGFP-positive cells was determined after 3-5 days. As shown in FIG. 7,each of the RHO 1-2 meganucleases tested preferentially cleaved the P23Hrecognition sequence relative to the RHO 3-4 recognition sequence, asevidenced by higher percentages of GFP-positive RHO 1-2 cells than RHO3-4 cells (see, FIG. 7A-7E). In subsequent experiments, it was shownthat recombinant meganucleases could be developed that preferentiallytarget and cleave the wild-type RHO 3-4 recognition sequence (SEQ IDNO:5). This set of meganucleases, when evaluated using the CHO cellreporter assay described above, were found to yield a higher percentageof GFP-positive RHO 3-4 cells than RHO 1-2 cells (see, FIG. 7F).

3. Conclusions

We have demonstrated that RHO meganucleases encompassed by the inventionpreferentially cleave the P23H recognition sequence of SEQ ID NO:1relative to the corresponding wild-type RHO 3-4 recognition sequence(SEQ ID NO:5).

Example 3 Generation and Expression of Recombinant AAV Vectors forExpressing Recombinant Meganucleases 1. Recombinant AAV Vectors forExpressing RHO 1-2 Meganucleases

Recombinant AAV vectors were designed to express RHO 1-2 meganucleasesin CHO reporter cells. As shown in FIG. 8A, the recombinant AAV vectorscomprise, from 5′ to 3′, a first inverted terminal repeat (ITR), a CMVpromoter operably linked to a nucleotide sequence encoding ameganuclease, and a second inverted terminal repeat. In this study, acoding sequence for the RHO-1/2-L2-49 meganuclease was incorporated intothe recombinant AAV vector.

2. Expression of a RHO 1-2 Meganuclease Following Recombinant AAVTransduction

A recombinant AAV vector (rAAV-RHO-1/2) was prepared for expression ofthe RHO-1/2-L2-49 meganuclease in RHO 1-2 cells using a standardtriple-transfection protocol in HEK-293 cells (Drittanti et al. (2001),J Gene Med. 3:59-71). RHO 1-2 CHO reporter cells were transduced withthe rAAV-RHO-1/2 vector at three concentrations of virus. At 24 hourspost-transduction, cells were lysed and analyzed by Western blot using apolyclonal meganuclease-specific antibody or a β-actin-specific controlantibody. As shown in FIG. 8B, RHO-1/2-L2-49 expression was observed atall three concentrations of virus.

3. Specificity of AAV-Delivered RHO 1-2 Meganuclease

The rAAV-RHO-1/2 vector was transduced into RHO 1-2 cells or RHO 3-4cells to demonstrate the specificity of AAV-delivered RHO-1/2-L2-49meganuclease for recognizing and cleaving the P23H recognition sequenceof SEQ ID NO:1 relative to the corresponding wild-type RHO 3-4recognition sequence (SEQ ID NO:5). As shown in FIG. 9, after three dayspost-transduction, the AAV-delivered RHO-1/2-L2-49 meganuclease induceda higher percentage of GFP-positive RHO 1-2 cells than RHO 3-4 cells atall concentrations of virus used, indicating that the meganucleasepreferentially recognizes and cleaves the mutant sequence.

4. Persistence of AAV-Delivered RHO 1-2 Meganuclease Expression

RHO 1-2 cells, transduced with the recombinant AAV vector encoding theRHO-1/2-L2-49 meganuclease or GFP, were treated with cycloheximide tohalt protein translation and determine the stability of the expressedproteins in cells. Cells were lysed at various times (0, 1.5, 6, and 22hours) post-cycloheximide treatment, and protein levels ofRHO-1/2-L2-49, GFP, and β-actin were determined by Western Blotanalysis. As shown in FIG. 10, RHO-1/2-L2-49 protein persistedsignificantly longer than GFP, with no reduction in meganuclease proteinapparent after 22 hours.

5. Conclusions

This study indicates that RHO 1-2 meganucleases encompassed by theinvention can be expressed in cells using recombinant AAV vectors, andthat such meganucleases can preferentially recognize and cleave the P23Hrecognition sequence of SEQ ID NO:1 relative to the correspondingwild-type RHO 3-4 sequence (SEQ ID NO:5). Furthermore, this studydemonstrates that RHO 1-2 meganuclease proteins are stable in cells whenexpressed using recombinant AAV vectors.

Example 4 Cleavage of RHO 1-2 Recognition Sequence in Reporter Cellswith RHO 1-2 Meganucleases Delivered by AAV 1. Production of RecombinantAAV Vectors

The purpose of this study was to demonstrate that RHO 1-2 meganucleasescould be expressed in mammalian cells by viral transduction, and tofurther demonstrate their ability to cleave the RHO 1-2 recognitionsequence.

For this experiment, two recombinant AAV2 vectors were produced by thetriple-transfection method previously described. The two donor plasmidswere pDS CMV RHO2_L3_59 (SEQ ID NO:248) and pDS CMV RHO2_L5_14 (SEQ IDNO:249), which encoded the RHO2-L3-59 and RHO2-L5-14 meganucleases,respectively (FIG. 11). These plasmids comprise a CMV promoter andenhancer driving expression of the nuclease, an SV40 polyA sequence, AAVITR (inverted terminal repeat) sequences required for genomereplication, and are suitable for the production of self-complimentaryAAV genomes (scAAV). The capsid from AAV serotype 2 was used to generatethese vectors. Recombinant scAAV was produced using thetriple-transfection method in HEK 293 cells and purified over a CsClgradient. Viral titer was determined by RT-PCR and slot-blot methods.

2. Transduction of Reporter Cells and Analysis of Cleavage Efficiency

To determine whether these AAV particles could deliver functional RHO1-2 meganucleases capable of recognizing and specifically cleaving theRHO 1-2 recognition sequence (SEQ ID NO:1) and not the RHO 3-4recognition sequence (SEQ ID NO:5), they were tested in previouslydescribed CHO reporter lines (see WO2012/167192). To perform the assay,a pair of CHO cell reporter lines were produced which carried anon-functional Green Fluorescent Protein (GFP) gene expression cassetteintegrated into the genome of the cell. The GFP gene in each cell linewas interrupted by either the RHO 1-2 or RHO 3-4 recognition sequencesuch that intracellular cleavage of the recognition sequence by ameganuclease would stimulate a homologous recombination event resultingin a functional GFP gene. CHO reporter cells comprising the RHO 1-2recognition sequence are referred to in this experiment as “P23H RHOcells.” CHO reporter cells comprising the RHO 3-4 recognition sequenceare referred to in this experiment as “WT RHO cells.” Wild-type controlcells that do not harbor the GFP reporter cassette are referred to inthis experiment as “CHO K cells.”

WT RHO cells, P23H RHO cells, or wild-type CHO K cells (negativecontrol) were infected with recombinant scAAV described above. Reportercells were infected at three MOIs: 1×10⁸, 1×10⁹ or 2×10⁹ (“low,”“medium,” and “high,” respectively). At 48 hours post-infection, cellswere evaluated by flow cytometry to determine the percentage ofGFP-positive cells compared to wild-type control CHO cells. As shown inFIG. 12, control wild-type CHO cells infected with either the RHO2-L3-59or RHO2-L5-14 scAAV provided background GFP expression, less than 1%.Cells harboring the wild-type RHO 3-4 target (“WT RHO cells”) did notdisplay GFP expression significantly higher than the control cells,indicating that the RHO 1-2 meganucleases are not capable of cleavingthe RHO 3-4 recognition sequence. In P23H RHO cells, both the RHO2-L3-59and RHO2-L5-14 meganucleases resulted in significant GFP expression in adose-dependent manner, with levels approaching 10% GFP+ in the highestdose.

Western blot analysis was used to confirm both GFP and meganucleaseexpression in the P23H RHO cells. Whole cell lysates were prepared onlyfrom P23H RHO and WT RHO cells infected with the three different dosesof scAAV. Lysates for duplicates were harvested to provide a measure ofreproducibility. Equivalent amounts of lysates (determined by proteinconcentration) were resolved by SDS-PAGE, transferred to a membrane andprobed using antibodies against either GFP, the I-CreI meganuclease, orfor loading control, β-actin. RHO 1-2 meganucleases are detectable withthe polyclonal antibody against I-CreI.

As shown in FIG. 13, western blot analysis clearly demonstratesdetection of GFP in a dose-dependent manner, confirming the flowcytometry data shown in FIG. 12 and described above. Western blotanalysis also shows expression of the RHO 1-2 meganucleases, also in adose-dependent manner. The blot does suggest that RHO2-L5-14 expressedto higher levels than RHO2-L3-59. β-actin levels were consistent,indicating proper gel loading.

3. Conclusions

Together, these data demonstrate recombinant scAAV carrying expressioncassettes for RHO 1-2 meganucleases of the invention are able to infectCHO reporter lines, resulting in expression of the RHO 1-2 meganucleaseswhich are then able to specifically cleave the P23H RHO 1-2 recognitionsequence (SEQ ID NO:1) and not the WT RHO 3-4 recognition

Example 5 In Vivo Cleavage of P23H Allele in Mouse Model of RP 1.Production of Recombinant AAV Vectors and Sub-Retinal Delivery to MouseEye

The purpose of this study was to determine whether RHO 1-2 meganucleasesof the invention could target and cleave the RHO 1-2 recognitionsequence in vivo within photoreceptor cells of a mouse retina.

Recombinant scAAV were prepared using the triple-transfection method asdescribed above and were tested in a murine model of retinitispigmentosa. In this model, transgenic mice carry a single copy of thehuman P23H mutant RHO gene in addition to the endogenous murine RHOalleles. While these mice do not exhibit a retinitis pigmentosaphenotype, they are useful for molecular analysis of in vivo cleavage ofthe RHO 1-2 recognition sequence. Since RHO2-L5-14 performed better inthe GFP reporter CHO lines, only scAAV encoding RHO2-L5-14 was used toinfect mice (though the present experiments could be performed using theRHO2-L3-59 meganuclease, including the production of an scAAV using thedonor plasmid set forth in SEQ ID NO:280). The pDS GRK1 RHO2_L5_14 donorplasmid (SEQ ID NO: 281) used in the scAAV triple transfection protocolis illustrated in FIG. 14. As shown, the RHO2-L5-14 meganuclease wasunder the control of a rod cell-specific GRK1 promoter. As a control, arecombinant scAAV encoding a GFP expression cassette was also prepared.The capsid from AAV serotype 5 was used to generate these recombinantscAAV vectors.

One of the P23H transgenic mice was used to test whether sub-retinalinjection of an AAV encoding a RHO2-L5-14 expression cassette couldresult in cleavage at the RHO 1-2 recognition sequence. Briefly, atpostnatal day 30, a mouse was placed under general anesthesia byintraperitoneal injection of Ketamine 100 mg/kg and Xylazine 10 mg/kg.Pupils were dilated with Tropicamide (0.5%)) and 1% Proparacaine. Underan ophthalmic surgery microscope, with a 30-gauge needle a smallincision was made through the cornea adjacent to the limbus. A 33-gaugeblunt needle fitted to a Hamilton syringe was inserted through theincision. All injections were made sub-retinally within the nasalquadrant of the retina. The mouse received 1 μL of scAAV encodingRHO2-L5-14 in one eye and 1 μL of scAAV encoding GFP in the other eye(both viral preparations were at a concentration of 7×10¹²particles/mL). Visualization during injection was aided by the additionof fluorescein to the vector suspensions. Fundus and OCT examinationwere performed to confirm the successful sub-retinal delivery.

The mouse was euthanized with isoflurane and eyes were enucleated at 30days post-injection. The retinas were carefully dissected free from theother ocular tissues under surgical microscope. DNA was isolated fromdissected retinas by using DNA isolation kit (Qiagen). Briefly, retinaswere digested with lysis buffer containing protease K at 55° C. for 2hrs. After lysis, crude extract was passed through the column and boundDNA washed several times. The DNA was eluted and concentration wasestimated by NanoDrop (Thermo).

To determine the presence and relative frequency of mutations at the RHO1-2 recognition sequence, the RHO 1-2 locus was PCR-amplified andsubjected to deep sequencing analysis. Briefly, PCR primers weredesigned to amplify a ˜200 bp region spanning the RHO 1-2 recognitionsequence and gel-purified PCR bands were subjected to deep sequencinganalysis using an Illumina MiSeq instrument.

2. Results

Deep sequencing of the PCR bands provided over 5×10⁵ sequences persample. Retinas injected with scAAV encoding GFP showed about 4×10³sequences with indels (insertions or deletions) at the RHO 1-2recognition sequence, establishing the background at 0.54%. In DNAisolated from a mouse retina that had been injected with AAV encodingthe RHO2-L5-14 meganuclease, indels were detected at 3.92%,approximately 7-fold higher than background (Table 3). In otherexperiments (data not shown), control mice showed a lower frequency ofindels, typically around 0.01%.

TABLE 3 Sequences with Sequences with indels no indels % Indel AAV-GFP4155 766145 0.54 AAV-RHO 1-2L5.14 22330 547763 3.92

3. Conclusions

The deep sequencing data demonstrates that sub-retinal injection with anAAV encoding the RHO2-L5-14 meganuclease resulted in mutation of theP23H RHO 1-2 recognition sequence in a murine model of retinitispigmentosa following cleavage and non-homologous end joining.

Example 6 In Vivo Expression of RHO 1-2 Meganucleases in RetinalCells 1. Western Blot Analysis of Retinal Cells

To confirm that the RHO2-L5-14 meganuclease could be expressed in theretinal cells of wild-type mice following AAV delivery, five wild-typemice were administered scAAV encoding RHO2-L5-14 by sub-retinalinjection as described above. Both the OS (left) and OD (right) eyeswere infected. 30 days following injection, retinas were dissected,whole cell lysates were prepared and western blot analysis wasperformed, using the anti-I-CreI antibody as described above.

Western blot analysis showed that in most retinas, RHO2L-5-14 expressionis readily detected (FIG. 15, lanes 3, 5, 7 and 10). In other retinas,expression was considerably less, but still detectable (FIG. 15, lanes1, 4, and 6). The remaining retinas had almost undetectable levels ofexpression (FIG. 15, lanes 2, 8 and 9). Sub-retinal injection relies onprecise delivery to a very small area behind the eye. Although thisprocedure is fairly common in adult patients, it is not a common labpractice in mouse models. Therefore, the difference in expression wasattributed to the difficulty of murine sub-retinal injections.

2. Conclusions

Western blot analysis demonstrated that scAAV encoding RHO2L-5-14delivered sub-retinally to mice resulted in expression of the IMO 1-2meganuclease. Taken together with indel data from a murine model for thehuman P23H RHO gene, these data suggest that AAV delivery of a RHO 1-2meganuclease is effective in causing deletions in the P23H RHO allele.

1.-35. (canceled)
 36. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a polynucleotide comprising anucleic acid sequence encoding an engineered meganuclease thatrecognizes and cleaves a P23H recognition sequence consisting of SEQ IDNO: 1, wherein said engineered meganuclease comprises a first subunitand a second subunit, and wherein said first subunit binds to a firstrecognition half-site of said P23H recognition sequence and comprises afirst hypervariable (HVR1) region, and wherein said second subunit bindsto a second recognition half-site of said P23H recognition sequence andcomprises a second hypervariable (HVR2) region, and wherein saidengineered meganuclease comprises an amino acid sequence having at least97% sequence identity to SEQ ID NO:7.
 37. The pharmaceutical compositionof claim 36, wherein said pharmaceutical composition comprises arecombinant adeno-associated viral (AAV) vector comprising saidpolynucleotide, wherein said nucleic acid sequence encoding saidengineered meganuclease is operably linked to a promoter.
 38. Thepharmaceutical composition of claim 37, wherein said recombinant AAVvector is serotype
 5. 39. The pharmaceutical composition of claim 37,wherein said recombinant AAV vector is serotype
 2. 40. Thepharmaceutical composition of claim 37, wherein said recombinant AAVvector is a self-complementary AAV vector.
 41. The pharmaceuticalcomposition of claim 36, wherein said first subunit comprises an aminoacid sequence having at least 97% sequence identity to residues 198-344of SEQ ID NO: 7 and wherein said second subunit comprises an amino acidsequence having at least 97% sequence identity to residues 7-153 of SEQID NO:
 7. 42. The pharmaceutical composition of claim 36, wherein saidfirst subunit comprises residues 198-344 of SEQ ID NO:
 7. 43. Thepharmaceutical composition of claim 36, wherein said second subunitcomprises residues 7-153 of SEQ ID NO:
 7. 44. The pharmaceuticalcomposition of claim 36, wherein said engineered meganuclease comprisesthe amino acid sequence of SEQ ID NO:
 7. 45. The pharmaceuticalcomposition of claim 36, wherein said engineered meganucleasepreferentially recognizes and cleaves a P23H recognition sequenceconsisting of SEQ ID NO: 1 relative to a recognition sequence consistingof SEQ ID NO:
 5. 46. The pharmaceutical composition of claim 37, whereinsaid promoter is a retina cell-specific promoter.
 47. The pharmaceuticalcomposition of claim 37, wherein said promoter is a rod photoreceptorcell-specific promoter.
 48. The pharmaceutical composition of claim 37,wherein said promoter is a human G-protein-coupled receptor proteinkinase 1 (GRK1) promoter.
 49. The pharmaceutical composition of claim37, wherein said recombinant AAV vector is serotype 5 and wherein saidrecombinant AAV vector is a self-complementary AAV vector.
 50. Thepharmaceutical composition of claim 37, wherein said recombinant AAVvector is serotype 5 and wherein said nucleic acid sequence encodingsaid engineered meganuclease is operably linked to a human GRK1promoter.
 51. The pharmaceutical composition of claim 37, wherein saidrecombinant AAV vector is serotype 5, and wherein said nucleic acidsequence encoding said engineered meganuclease is operably linked to ahuman GRK1 promoter, and wherein said recombinant AAV vector is aself-complementary AAV vector.
 52. The pharmaceutical composition ofclaim 37, wherein said recombinant AAV vector is serotype 5, whereinsaid promoter is a human GRK1 promoter, and wherein said engineeredmeganuclease comprises the amino acid sequence of SEQ ID NO:
 7. 53. Thepharmaceutical composition of claim 37, wherein said recombinant AAVvector is serotype 5, wherein said promoter is a human GRK1 promoter,wherein said engineered meganuclease comprises the amino acid sequenceof SEQ ID NO: 7, and wherein said recombinant AAV vector is aself-complementary AAV vector.